ABSTRACT
OBJECTIVE@#To explore the effect of shikonin on the recovery of nerve function after acute spinal cord injury(SCI) in rats.@*METHODS@#96 male Sprague-Dawley(SD)rats were divided into 4 groups randomly:sham operation group (Group A), sham operation+shikonin group (Group B), SCI+ DMSO(Group C), SCI+shikonin group (Group D).The acute SCI model of rats was made by clamp method in groups C and D . After subdural catheterization, no drug was given in group A. rats in groups B and D were injected with 100 mg·kg-1 of shikonin through catheter 30 min after modeling, and rats in group C were given with the same amount of DMSO, once a day until the time point of collection tissue. Basso-Beattie-Bresnahan(BBB) scores were performed on 8 rats in each group at 6, 12, and 3 d after moneling, and oblique plate tests were performed on 1, 3, 7 and 14 d after modeling, and then spinal cord tissues were collected. Eight rats were intraperitoneally injected with propidine iodide(PI) 1 h before sacrificed to detection PI positive cells at 24 h in each group. Eight rats were sacrificed in each group at 24 h after modeling, the spinal cord injury was observed by HE staining.The Nissl staining was used to observe survivor number of nerve cells. Western-blot technique was used to detect the expression levels of Bcl-2 protein and apoptosis related protein RIPK1.@*RESULTS@#After modeling, BBB scores were normal in group A and B, but in group C and D were significantly higher than those in group A and B. And the scores in group D were higher than those in group C in each time point (P<0.05). At 12 h after modeling, the PI red stained cells in group D were significantly reduced compared with that in group C, and the disintegration of neurons was alleviated(P<0.05). HE and Nissl staining showed nerve cells with normal morphology in group A and B at 24h after operation. The degree of SCI and the number of neuronal survival in group D were better than those in group C, the difference was statistically significant at 24h (P<0.05). The expression of Bcl-2 and RIPK1 proteins was very low in group A and B;The expression of RIPK1 was significantly increased in Group C and decreased in Group D, with a statistically significant difference (P<0.05);The expression of Bcl-2 protein in group D was significantly higher than that in group C (P<0.05).@*CONCLUSION@#Shikonin can alleviate the pathological changes after acute SCI in rats, improve the behavioral score, and promote the recovery of spinal nerve function. The specific mechanism may be related to the inhibition of TNFR/RIPK1 signaling pathway mediated necrotic apoptosis.
Subject(s)
Animals , Male , Rats , Dimethyl Sulfoxide/metabolism , Naphthoquinones , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats, Sprague-Dawley , Spinal Cord/metabolism , Spinal Cord Injuries/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolismABSTRACT
Objective To establish U251 cells with inhibited expression of interferon-γ inducible protein 30 (IFI30), and to investigate the effect of IFI30 on cell biological function as well as its underlying mechanism. Methods Three knockdown sequences which target IFI30 were designed online and 3 small interfering RNAs (siRNA) were synthesized. After transfection, the inhibition efficiency was detected by real-time quantitative PCR. The siRNA sequence with the highest inhibition efficiency was selected to create short hairpin RNA (shRNA) plasmids. The recombinant plasmids and packaging plasmids were co-transfected into HEK293T cells to prepare lentivirus. The glioma U251 cells were transfected with lentivirus, and the positive cells were screened by puromycin. CCK-8 assay, 5-ethyl-2'-deoxyuridine (EdU) and colony formation assays were used to analyze cell proliferation; the flow cytometry was used to analyze cell cycle and apoptosis; the TranswellTM assay was used to detect cell invasion; the wound-healing assay was employed to detect cell migration, and western blot analysis to detect the protein expresison of cyclin D1, B-cell lymphoma factor 2 (Bcl2), epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), signal transducer and activator of transcription 1 (STAT1). Results The sequence which effectively target IFI30 was screened and U251 cell line capable of inhibiting the IFI30 expression was successfully established. When IFI30 expression was knocked down, the proliferation of U251 cells was inhibited, along with increased ratio of cells in the phase G0/G1, the decreased phase S, the increased rate of cell apoptosis. The cell invasion and migration capabilities was also reduced. The decreased expression of cyclin D1, Bcl2 and N-cadherin were observed in U251 cells, and the expression of E-cadherin and the phosphorylation of STAT1 were found increased. Conclusion Knockdown of IFI30 inhibits the proliferation, invasion and migration of human glioma cell U251 and promotes its apoptosis by activating STAT1.
Subject(s)
Humans , Cyclin D1/genetics , HEK293 Cells , Interferon-gamma , RNA, Small Interfering , Apoptosis/genetics , Cadherins , Cell Proliferation/genetics , Glioma/genetics , Proto-Oncogene Proteins c-bcl-2 , Oxidoreductases Acting on Sulfur Group Donors , STAT1 Transcription Factor/geneticsABSTRACT
PURPOSE@#The incidence of heatstroke (HS) is not particularly high; however, once it occurs, the consequences are serious. It is reported that calcitonin gene-related peptide (CGRP) is protective against brain injury in HS rats, but detailed molecular mechanisms need to be further investigated. In this study, we further explored whether CGRP inhibited neuronal apoptosis in HS rats via protein kinase A (PKA)/p-cAMP response element-binding protein (p-CREB) pathway.@*METHODS@#We established a HS rat model in a pre-warmed artificial climate chamber with a temperature of (35.5 ± 0.5) °C and a relative humidity of 60% ± 5%. Heatstress was stopped once core body temperature reaches above 41 °C. A total of 25 rats were randomly divided into 5 groups with 5 animals each: control group, HS group, HS+CGRP group, HS+CGRP antagonist (CGRP8-37) group, and HS+CGRP+PKA/p-CREB pathway blocker (H89) group. A bolus injection of CGRP was administered to each rat in HS+CGRP group, CGRP8-37 (antagonist of CGRP) in HS+CGRP8-37 group, and CGRP with H89 in HS+CGRP+H89 group. Electroencephalograms were recorded and the serum concentration of S100B, neuron-specific enolase (NSE), neuron apoptosis, activated caspase-3 and CGRP expression, as well as pathological morphology of brain tissue were detected at 2 h, 6 h, and 24 h after HS in vivo. The expression of PKA, p-CREB, and Bcl-2 in rat neurons were also detected at 2 h after HS in vitro. Exogenous CGRP, CGRP8-37, or H89 were used to determine whether CGRP plays a protective role in brain injury via PKA/p-CREB pathway. The unpaired t-test was used between the 2 samples, and the mean ± SD was used for multiple samples. Double-tailed p < 0.05 was considered statistically significant.@*RESULTS@#Electroencephalogram showed significant alteration of θ (54.50 ± 11.51 vs. 31.30 ± 8.71, F = 6.790, p = 0.005) and α wave (16.60 ± 3.21 vs. 35.40 ± 11.28, F = 4.549, p = 0.020) in HS group compared to the control group 2 h after HS. The results of triphosphate gap terminal labeling (TUNEL) showed that the neuronal apoptosis of HS rats was increased in the cortex (9.67 ± 3.16 vs. 1.80 ± 1.10, F = 11.002, p = 0.001) and hippocampus (15.73 ± 8.92 vs. 2.00 ± 1.00, F = 4.089, p = 0.028), the expression of activated caspase-3 was increased in the cortex (61.76 ± 25.13 vs. 19.57 ± 17.88, F = 5.695, p = 0.009) and hippocampus (58.60 ± 23.30 vs. 17.80 ± 17.62, F = 4.628, p = 0.019); meanwhile the expression of serum NSE (5.77 ± 1.78 vs. 2.35 ± 0.56, F = 5.174, p = 0.013) and S100B (2.86 ± 0.69 vs. 1.35 ± 0.34, F = 10.982, p = 0.001) were increased significantly under HS. Exogenous CGRP decreased the concentrations of NSE and S100B, and activated the expression of caspase-3 (0.41 ± 0.09 vs. 0.23 ± 0.04, F = 32.387, p < 0.001) under HS; while CGRP8-37 increased NSE (3.99 ± 0.47 vs. 2.40 ± 0.50, F = 11.991, p = 0.000) and S100B (2.19 ± 0.43 vs. 1.42 ± 0.30, F = 4.078, p = 0.025), and activated the expression caspase-3 (0.79 ± 0.10 vs. 0.23 ± 0.04, F = 32.387, p < 0.001). For the cell experiment, CGRP increased Bcl-2 (2.01 ± 0.73 vs. 2.15 ± 0.74, F = 8.993, p < 0.001), PKA (0.88 ± 0.08 vs. 0.37 ± 0.14, F = 20.370, p < 0.001), and p-CREB (0.87 ± 0.13 vs. 0.29 ± 0.10, F = 16.759, p < 0.001) levels; while H89, a blocker of the PKA/p-CREB pathway reversed the expression.@*CONCLUSIONS@#CGRP can protect against HS-induced neuron apoptosis via PKA/p-CREB pathway and reduce activation of caspase-3 by regulating Bcl-2. Thus CGRP may be a new target for the treatment of brain injury in HS.
Subject(s)
Animals , Rats , Apoptosis , Brain Injuries/pathology , Calcitonin Gene-Related Peptide/metabolism , Caspase 3 , Isoquinolines , Proto-Oncogene Proteins c-bcl-2 , Rats, Sprague-Dawley , Sulfonamides , Heat Stroke/pathologyABSTRACT
OBJECTIVE@#To investigate the effects of asperuloside on cervical cancer based on endoplasmic reticulum (ER) stress and mitochondrial pathway.@*METHODS@#Different doses (12.5-800 µg/mL) of asperuloside were used to treat cervical cancer cell lines Hela and CaSki to calculate the half maximal inhibitory concentration (IC50) of asperuloside. The cell proliferation was analyzed by clone formation assay. Cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by flow cytometry. The protein expressions of cleaved-caspase-3, Bcl-2, Bax, Cyt-c, cleaved-caspase-4 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot. And the inhibitor of ER stress, 4-phenyl butyric acid (4-PBA) was used to treat cervical cancer cells to further verify the role of ER stress in the apoptosis of cervical cancer cells induced by asperuloside.@*RESULTS@#Asperuloside of 325, 650, and 1300 µg/mL significantly inhibited the proliferation and promoted apoptosis of Hela and CaSki cells (P<0.01). All doses of asperuloside significantly increased intracellular ROS levels, reduced mitochondrial membrane potential, significantly reduced Bcl-2 protein expression level, and increased Bax, Cyt-c, GRP78 and cleaved-caspase-4 expressions (P<0.01). In addition, 10 mmol/L 4-PBA treatment significantly promoted cell proliferation and reduced apoptosis (P<0.05), and 650 µg/mL asperuloside could reverse 4-PBA-induced increased cell proliferation, decreased apoptosis and cleaved-caspase-3, -4 and GRP78 protein expressions (P<0.05).@*CONCLUSION@#Our study revealed the role of asperuloside in cervical cancer, suggesting that asperuloside promotes apoptosis of cervical cancer cells through ER stress-mitochondrial pathway.
Subject(s)
Female , Humans , Uterine Cervical Neoplasms/metabolism , Caspase 3/metabolism , bcl-2-Associated X Protein/metabolism , Reactive Oxygen Species/metabolism , Endoplasmic Reticulum Chaperone BiP , HeLa Cells , Proto-Oncogene Proteins c-bcl-2/metabolism , Apoptosis , Endoplasmic Reticulum Stress , Cell Line, TumorABSTRACT
Objective: To investigate the effect of hsa_circ_0000392 (circ_0000392) on the radiosensitivity of cervical cancer cells and explore its potential mechanism. Methods: Cervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected. According to the patients' response to radiotherapy, the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues. The expressions of circ_0000392, miR-145-5p, and CRKL in radiation-sensitive, radiation-resistant cervical cancer tissues and Hela, SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. SiRNA circ_0000392, miR-145-5p mimic, miR-145-5p inhibitor, pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells, respectively. After radiation induction, the survival fraction of cells was detected by clone formation assay, apoptosis was detected by flow cytometry, and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot. The targeting relationship between circ_0000392, miR-145-5p and CRKL was verified by dual luciferase reporter gene assay. The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice. Results: circ_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer, while miR-145-5p was downregulated. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ 6 Gy group were (78.67±10.97) and (71.00±9.54), respectively, which were lower than those in si-Ctrl+ 6 Gy group [(176.00±22.27) and (158.33±17.56), respectively]. The apoptosis rates were (41.55±3.40)% and (31.41±3.29)%, respectively, which were higher than those in si-Ctrl+ 6 Gy group [(15.91±1.37)% and (13.70±1.89)%, P<0.05]. The protein expression of Bax was higher than that of si-Ctrl+ 6 Gy group, and the protein expressions of Bcl2 was lower than those of si-Ctrl+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ miR-145-5p inhibitor+ 6 Gy group were (171.33±25.01) and (137.00±21.66), higher than those in si-circ_0000392#1+ inhibitor NC+ 6 Gy group [(84.67±17.79) vs (71.00±11.00), P<0.05]. The apoptosis rates were (17.41±2.58) % and (15.96±1.25) %, lower than those of si-circ_0000392 #1+ inhibitor NC+ 6 Gy [(40.29±2.92)% and (30.82±2.34)%, respectively, P<0.05]. The expression of Bax protein was lower than that of si-circ_0000392#1+ inhibitor NC+ 6 Gy group, and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+ inhibitor NC+ 6 Gy group. Circ_0000392 can target miR-145-5p, and CRKL is the downstream target gene of miR-145-5p. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ 6 Gy group were (74.33±10.02) and (66.00±12.17), respectively, which were lower than those of mimic NC+ 6 Gy group [(197.67±17.21) vs (157.67±11.59), respectively, P<0.05]. The apoptosis rates were (45.58±2.16)% and (32.10±3.55)%, higher than those of mimic NC+ 6 Gy group [(15.85±2.45)% and (13.99±1.69)%, respectively, P<0.05]. The expression of Bax protein was higher than that of the mimic NC+ 6 Gy mimic group, and the expression of Bcl2 protein was lower than that of the mimic NC+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ pcDNA-CRKL+ 6 Gy group were (158.00±15.88) and (122.33±13.65), respectively, which were higher than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(71.33±8.02) vs (65.67±12.22), P<0.05]. The apoptosis rates were (19.50±3.45)% and (17.04±0.94)%, respectively, which were lower than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(44.33±2.36)% and (32.05±2.76)%, respectively, P<0.05]. The expression of Bax protein was lower than that of miR-145-5p mimic+ pcDNA group+ 6 Gy group, and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+ pcDNA+ 6 Gy group. Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight (P<0.05). The relative mRNA expression levels of circ_0000392, miR-145-5p and CRKL and the relative protein expression levels of CRKL, Bcl-2 and p-ERK1/2 were decreased, while the relative expression level of Bax protein was increased (P<0.05). Conclusion: Circ_0000392 could enhance the radiosensitivity of cervical cancer cells, and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p, which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.
Subject(s)
Animals , Mice , Female , Humans , Uterine Cervical Neoplasms/radiotherapy , bcl-2-Associated X Protein/genetics , Mice, Nude , Proto-Oncogene Proteins c-bcl-2/genetics , Apoptosis , MicroRNAs/genetics , Cell Proliferation , Cell Line, TumorABSTRACT
OBJECTIVE@#To investigate the effect of microRNA-509-3p (miR-509-3p) on the apoptosis of atherosclerotic vascular endothelial cells.@*METHODS@#Mouse aortic endothelial cells (MAECs) were divided into normal control group, oxidized low-density lipoprotein (ox-LDL) group, miR-509-3p overexpression group, miR-509-3p overexpression control group, miR-509-3p inhibitor + ox-LDL group, and miR-509-3p inhibitor control + ox-LDL group. MAEC were induced with 100 mg/L ox-LDL for 24 hours, and then transfected with miR-509-3p overexpression/inhibitor and corresponding control for 48 hours. The miR-509-3p expression in MAECs exposed to ox-LDL was detected using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Flow cytometry was used to detect the level of apoptosis, and cell counting kit (CCK-8) was used to detect the proliferation activity of MAECs. The direct gene targets of miR-509-3p were predicted using bioinformatics analyses and confirmed using a dual luciferase reporter assay. The expression of Bcl-2 mRNA and protein was detected by RT-qPCR and Western blotting, respectively.@*RESULTS@#Compared with the normal control group, miR-509-3p was significantly upregulated in ox-LDL-stimulated MAECs (1.68±0.85 vs. 1.00±0.30, t = 2.398, P < 0.05). After transfection of MAECs with miR-509-3p overexpression, the luciferase activity of the BCL2 3'UTR WT reporter gene was significantly lower than that of miR-509-3p overexpression control group (0.83±0.06 vs. 1.00±0.07, t = 4.531, P = 0.001). The luciferase activity of the BCL2 3'-UTR mutant (MUT) reporter gene was not significantly different from that of miR-509-3p overexpression control group (0.94±0.05 vs. 1.00±0.08, t = 1.414, P = 0.188). Compared with the normal control group and miR-509-3p mimics control group, the cell proliferation activity was decreased [(0.60±0.06)% vs. (1.00±0.09)%, (0.89±0.04)%, both P < 0.01], the percentage of apoptotic cells were increased [(23.46±2.02)% vs. (7.66±1.52)%, (10.40±0.78)%, both P < 0.05], and the mRNA and protein expression of Bcl-2 were significantly downregulated (Bcl-2 mRNA: 0.52±0.13 vs. 1.00±0.36, 1.10±0.19, Bcl-2 protein: 0.42±0.07 vs. 1.00±0.11, 0.93±0.10, both P < 0.01) in miR-509-3p overexpression group. Compared with the ox-LDL group, inhibition of miR-509-3p expression could increase the proliferation activity of MAECs induced by ox-LDL [(0.64±0.35)% vs. (0.34±0.20%)%, P < 0.05], and reduce the apoptosis rate [(13.59±2.22)% vs. (29.84±5.19)%, P < 0.01], and up-regulated the expression of Bcl-2 mRNA and protein in MAECs induced by ox-LDL (Bcl-2 mRNA relative expression: 0.82±0.09 vs. 0.52±0.10, Bcl-2 protein relative expression: 0.83±0.17 vs. 0.40±0.07, both P < 0.05).@*CONCLUSIONS@#Bcl-2 was one of the target genes of miR-509-3p. miR-509-3p can reduce the proliferation activity of endothelial cells, reduce the expression of Bcl-2, and promote cell apoptosis, thereby promoting the occurrence and development of atherosclerosis. Inhibition of miR-509-3p expression may be a potential therapeutic target for atherosclerosis.
Subject(s)
Animals , Mice , Humans , Endothelial Cells , MicroRNAs/metabolism , Signal Transduction , Lipoproteins, LDL/metabolism , Apoptosis , RNA, Messenger/metabolism , Proto-Oncogene Proteins c-bcl-2/pharmacology , Atherosclerosis/metabolism , Luciferases/pharmacology , Cell Proliferation , Human Umbilical Vein Endothelial CellsABSTRACT
Objective: To summarize the pathological diagnosis, clinical features, treatment methods and outcomes of pediatric-type follicular lymphoma (PTFL). Methods: Clinical data including the pathology, clinical features, treatment methods, and follow-up results of 9 PTFL patients admitted to Henan Cancer Hospital from February 2017 to February 2023 were analyzed retrospectively. Results: The age of onset in 9 children was 6 to 18 years, all the patients were males. The clinical manifestation was local painless lymph node enlargement in the head and neck, with a stage of Ⅰ-Ⅱ. The histomorphological characteristics of PTFL were similar to those of classic follicular lymphoma (FL). The germinal center of most follicles were enlarged, the mantle zone disappeared, centroblasts were easily visible, and the histological grade were mostly grade Ⅲ, which may be accompanied by the "starry sky" phenomenon. Monoclonal peaks can be seen in B cell clonal rearrangements (BCR). Immunohistochemistry (IHC) showed CD20 positive, CD10 positive, Bcl-6 positive, Bcl-2 negative, C-myc negative, and Ki-67 was 70%-95%. Fluorescence in situ hybridization (FISH) test was negative for t (14, 18), Bcl-2 translocation, and C-myc translocation. Six cases underwent surgical resection, and 3 cases underwent surgical resection combined with chemotherapy. Up to February 2023, with a follow-up time of 45 to 72 months, all children survived without any recurrence and were in a complete remission state. Conclusions: PTFL is mainly characterized by adolescent male onset, with early clinical manifestations and pathological manifestations of high-level histological status, high proliferation index, and lack of t (14; 18)/Bcl-2 translocation and Bcl-2 expression. It is mainly treated by localized surgical excision and has a good prognosis.
Subject(s)
Child , Adolescent , Humans , Male , Female , Lymphoma, Follicular/pathology , Lymphoma, B-Cell/pathology , In Situ Hybridization, Fluorescence , Retrospective Studies , Proto-Oncogene Proteins c-bcl-2/geneticsABSTRACT
OBJECTIVE@#To observe the efficacy and safety of different induction regimens of same total dosage of azacitidine (Aza), including standard dose (standard dose group) and low-dose long-term (adjusted dose group), in the treatment of elderly acute myeloid leukemia (AML).@*METHODS@#A total of 103 elderly patients with AML (non-acute promyelocytic leukemia) from January 2020 to June 2021 were enrolled. Aza was administered at the standard dose of 75 mg/(m2·d) for 7 days in the standard dose group (50 cases), while at 100 mg/d for 7-12 days in the adjusted dose group (53 cases). The administration days in adjusted dose group was calculated based on the total standard dose of the patient's single course of treatment. The efficacy and safety between standard dose group and adjusted dose group were compared. Subgroup analysis were performed in the two groups for Aza alone, Aza combined with BCL-2 inhibitor, and Aza combined with low-dose chemotherapy for efficacy and safety.@*RESULTS@#There were no significant differences in overall response rate (ORR), incidence of adverse reaction, and 1-year overall survival (OS) rate between standard dose group and adjusted dose group (P >0.05). The ORR of combination was higher than that of Aza alone (P < 0.05), while there was no significant difference in ORR between Aza combined with BCL-2 inhibitor and Aza combined with low-dose chemotherapy (P >0.05). The combination of BCL-2 inhibitor did not increase the incidence of adverse reactions compared wtih Aza alone. There was a higher risk of myelosuppression and pulmonary infection with a combination of low-dose chemotherapy than with a combination of BCL-2 inhibitor and Aza alone (P <0.05). No significant difference was observed in 1-year OS between Aza alone, Aza combined with BCL-2 inhibitor, and Aza combined with low-dose chemotherapy (P >0.05).@*CONCLUSIONS@#Both two induction regimens can be used in elderly AML patients who cannot tolerate intensive chemotherapy with similar overall effectiveness and safety. Aza combined with low-dose chemotherapy may result in increased ORR and an increased incidence of serious adverse reactions, and may not result in longer survival compared with Aza alone. Aza combined with BCL-2 inhibitor not only has similar effect in complete remission, objective response rate, and OS compared with Aza combined with low-dose chemotherapy, but also has higher safety.
Subject(s)
Humans , Aged , Azacitidine/therapeutic use , Prospective Studies , Treatment Outcome , Antineoplastic Combined Chemotherapy Protocols , Leukemia, Myeloid, Acute/etiology , Proto-Oncogene Proteins c-bcl-2ABSTRACT
OBJECTIVE@#To investigate the effects of methionine restriction on proliferation, cell cycle and apoptosis of human acute leukemia cells.@*METHODS@#Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of methionine restriction on HL-60 and Jurkat cells proliferation. The effect of methionine restriction on cell cycle of HL-60 and Jurkat cells was examined by PI staining. Annexin V-FITC / PI double staining was applied to detect apoptosis of HL-60 and Jurkat cells following methionine restriction. The expression of cell cycle-related proteins cyclin B1, CDC2 and apoptosis-related protein Bcl-2 was evaluated by Western blot assay.@*RESULTS@#Methionine restriction significantly inhibited the proliferation of HL-60 and Jurkat cells in a time-dependent manner (HL-60: r =0.7773, Jurkat: r =0.8725), arrested the cells at G2/M phase (P < 0.001), and significantly induced apoptosis of HL-60 and Jurkat cells (HL-60: P < 0.001; Jurkat: P < 0.05). Furthermore, Western blot analysis demonstrated that methionine restriction significantly reduced the proteins expression of Cyclin B1 (P < 0.05), CDC2 (P < 0.01) and Bcl-2 (P < 0.001) in HL-60 and Jurkat cells.@*CONCLUSION@#Acute leukemia cells HL-60 and Jurkat exhibit methionine dependence. Methionine restriction can significantly inhibit the proliferation, promote cell cycle arrest and induce apoptosis of HL-60 and Jurkat cells, which suggests that methionine restriction may be a potential therapeutic strategy for acute leukemia.
Subject(s)
Humans , Cyclin B1/pharmacology , Cell Proliferation , Methionine/pharmacology , Cell Cycle , Apoptosis , Leukemia, Myeloid, Acute , Cell Division , Cell Cycle Proteins , Jurkat Cells , Proto-Oncogene Proteins c-bcl-2/metabolism , HL-60 CellsABSTRACT
OBJECTIVE@#To investigate the effect and underlying mechanism of hispidulin on the proliferation and apoptosis of leukemia K562 cells.@*METHODS@#K562 cells were cultured in vitro and treated with 0, 5, 25 or 100 μmol/L hispidulin for 24 h. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Western blot was used to assess the expression of Bax, Bcl-2 and interleukin (IL)-37 proteins. Bone marrow mononuclear cells were extracted from 17 chronic myeloid leukemia patients and 21 healthy individuals by Ficoll-Hypaque density gradient method, and the expression of IL-37 protein was measured by Western blot. K562 cells with IL-37 overexpression or knockdown were constructed, and then treated with 0 or 100 μmol/L hispidulin for 24 h. Cell proliferation, apoptosis and protein expression of Bax and Bcl-2 were determined in the same way as above.@*RESULTS@#After K562 cells were treated with hispidulin, the cell inhibition rate, apoptosis rate, and the protein expression of Bax and IL-37 were significantly increased (P <0.05), but the cell proliferation and expression of Bcl-2 protein were decreased (P <0.05). The expression of IL-37 protein in bone marrow mononuclear cells of the leukemia patient was 0.24±0.03, which was significantly lower than 0.91±0.05 of healthy controls (P <0.05). Overexpression of IL-37 significantly promoted inhibition rate, apoptosis rate, and expression of Bax protein in K562 cells (P <0.05), but suppressed the expression of Bcl-2 protein (P <0.05). In addition, knockdown of IL-37 could reverse the effects of hispidulin on proliferation and apoptosis of K562 cells.@*CONCLUSION@#Hispidulin inhibits the proliferation and induces apoptosis of leukemia K562 cells, which may be related to the up-regulation of IL-37 protein in cells.
Subject(s)
Humans , K562 Cells , bcl-2-Associated X Protein/pharmacology , Apoptosis , Leukemia , Proto-Oncogene Proteins c-bcl-2 , Cell ProliferationABSTRACT
OBJECTIVE@#To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma.@*METHODS@#Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR).@*RESULTS@#Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05).@*CONCLUSION@#Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.
Subject(s)
Humans , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Apoptosis Regulatory Proteins/immunology , Autophagy/immunology , bcl-2-Associated X Protein/immunology , Bortezomib/therapeutic use , Burkitt Lymphoma/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Therapy, Combination , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2 , Receptors, CXCR/immunology , RNA, Messenger , TOR Serine-Threonine Kinases , Xanthones/therapeutic useABSTRACT
OBJECTIVE@#To explore the mechanism of Radix Scrophulariae (RS) extracts in the treatment of hyperthyroidism rats by regulating proliferation, apoptosis, and autophagy of thyroid cell through the mammalian sterile 20-like kinase 1 (MST1)/Hippo pathway.@*METHODS@#Twenty-four rats were randomly divided into 4 groups according to a random number table: control, model group, RS, and RS+Hippo inhibitor (XMU-MP-1) groups (n=6 per group). Rats were gavaged with levothyroxine sodium tablet suspension (LST, 8 μ g/kg) for 21 days except for the control group. Afterwards, rats in the RS group were gavaged with RS extracts at the dose of 1,350 mg/kg, and rats in the RS+XMU-MP-1 group were gavaged with 1,350 mg/kg RS extracts and 1 mg/kg XMU-MP-1. After 15 days of administration, thyroid gland was taken for gross observation, and histopathological changes were observed by hematoxylin-eosin staining. The structure of Golgi secretory vesicles in thyroid tissues was observed by transmission electron microscopy. The expression of thyrotropin receptor (TSH-R) was observed by immunohistochemistry. Terminal-deoxynucleoitidyl transferase mediated nick end labeling assay was used to detect cell apoptosis in thyroid tissues. Real-time quantity primer chain reaction and Western blot were used to detect the expressions of MST1, p-large tumor suppressor gene 1 (LATS1), p-Yes1 associated transcriptional regulator (YAP), proliferating cell nuclear antigen (PCNA), G1/S-specific cyclin-D1 (Cyclin D1), B-cell lymphoma-2 (Bcl-2), Caspase-3, microtubule-associated proeins light chain 3 II/I (LC3-II/I), and recombinant human autophagy related 5 (ATG5). Thyroxine (T4) level was detected by enzyme-linked immunosorbent assay.@*RESULTS@#The thyroid volume of rats in the model group was significantly increased compared to the normal control group (P<0.01), and pathological changes such as uneven size of follicular epithelial cells, disorderly arrangement, and irregular morphology occurred. The secretion of small vesicles by Golgi apparatus was reduced, and the expressions of receptor protein TSH-R and T4 were significantly increased (P<0.01), while the expressions of MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 were significantly decreased (P<0.01). The expressions of Bcl-2, PCNA, and cyclin D1 were significantly increased (P<0.01). Compared with the model group, RS extracts reduced the volume of thyroid gland, improved pathological condition of the thyroid gland, promoted secretion of the secretory vesicles with double-layer membrane structure in thyroid Golgi, significantly inhibited the expression of TSH-R and T4 levels (P<0.01), upregulated MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 expressions (P<0.01), and downregulated Bcl-2, PCNA, and Cyclin D1 expressions (P<0.01). XMU-MP-1 inhibited the intervention effects of RS extracts (P<0.01).@*CONCLUSION@#RS extracts could inhibit proliferation and promote apoptosis and autophagy in thyroid tissues through MST1/Hippo pathway for treating hyperthyroidism.
Subject(s)
Rats , Humans , Animals , Hippo Signaling Pathway , Proliferating Cell Nuclear Antigen/metabolism , Cyclin D1/pharmacology , Caspase 3/metabolism , Protein Serine-Threonine Kinases/pharmacology , Apoptosis , Hyperthyroidism/drug therapy , Proto-Oncogene Proteins c-bcl-2/metabolism , Thyrotropin/pharmacology , Mammals/metabolismABSTRACT
This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.
Subject(s)
Male , Animals , Mice , Cisplatin/pharmacology , Carcinoma, Hepatocellular/genetics , MAP Kinase Signaling System , Beclin-1 , Apoptosis , Liver Neoplasms/genetics , Necrosis , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Line, Tumor , RNA, Messenger/metabolism , AutophagyABSTRACT
This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.
Subject(s)
Humans , Female , Breast Neoplasms/metabolism , MCF-7 Cells , Caspase 3/metabolism , Caspase 9/metabolism , Beclin-1/pharmacology , Apoptosis , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm , Cell ProliferationABSTRACT
This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.
Subject(s)
Rats , Animals , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Caspase 3/genetics , Tumor Necrosis Factor-alpha/metabolism , bcl-2-Associated X Protein/metabolism , Plant Bark , Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/chemically induced , Inflammation/drug therapy , Cytokines/metabolism , Proto-Oncogene Proteins c-bcl-2 , ApoptosisABSTRACT
This study aims to explore the neuroprotective mechanism of ginsenoside Re(GS-Re) on drosophila model of Parkinson's disease(PD) induced by rotenone(Rot). To be specific, Rot was used to induce PD in drosophilas. Then the drosophilas were grouped and respectively treated(GS-Re: 0.1, 0.4, 1.6 mmol·L~(-1); L-dopa: 80 μmol·L~(-1)). Life span and crawling ability of drosophilas were determined. The brain antioxidant activity [content of catalase(CAT), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD)], dopamine(DA) content, and mitochondrial function [content of adenosine triphosphate(ATP), NADH:ubiquinone oxidoreductase subunit B8(NDUFB8) Ⅰ activity, succinate dehydrogenase complex, subunit B(SDHB) Ⅱ activity] were detected by enzyme-linked immunosorbent assay(ELISA). The number of DA neurons in the brains of drosophilas was measured with the immunofluorescence method. The levels of NDUFB8 Ⅰ, SDHB Ⅱ, cytochrome C(Cyt C), nuclear factor-E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), B-cell lymphoma/leukemia 2(Bcl-2)/Bcl-2-assaciated X protein(Bax), and cleaved caspase-3/caspase-3 in the brain were detected by Western blot. The results showed that model group [475 μmol·L~(-1) Rot(IC_(50))] demonstrated significantly low survival rate, obvious dyskinesia, small number of neurons and low DA content in the brain, high ROS level and MDA content, low content of SOD and CAT, significantly low ATP content, NDUFB8 Ⅰ activity, and SDHB Ⅱ activity, significantly low expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax, large amount of Cyt C released from mitochondria to cytoplasm, low nuclear transfer of Nrf2, and significantly high expression of cleaved caspase-3/caspase-3 compared with the control group. GS-Re(0.1, 0.4, and 1.6 mmol·L~(-1)) significantly improved the survival rate of PD drosophilas, alleviated the dyskinesia, increased DA content, reduced the loss of DA neurons, ROS level, and MDA content in brain, improved content of SOD and CAT and antioxidant activity in brain, maintained mitochondrial homeostasis(significantly increased ATP content and activity of NDUFB8 Ⅰ and SDHB Ⅱ, significantly up-regulated expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax), significantly reduced the expression of Cyt C, increased the nuclear transfer of Nrf2, and down-regulated the expression of cleaved caspase-3/caspase-3. In conclusion, GS-Re can significantly relieve the Rot-induced cerebral neurotoxicity in drosophilas. The mechanism may be that GS-Re activates Keap1-Nrf2-ARE signaling pathway by maintaining mitochondrial homeostasis, improves antioxidant capacity of brain neurons, then inhibits mitochondria-mediated caspase-3 signaling pathway, and the apoptosis of neuronal cells, thereby exerting the neuroprotective effect.
Subject(s)
Animals , Reactive Oxygen Species/metabolism , Antioxidants/pharmacology , Oxidative Stress , NF-E2-Related Factor 2/metabolism , Caspase 3/metabolism , Parkinson Disease/genetics , bcl-2-Associated X Protein/metabolism , Neuroprotective Agents/pharmacology , Kelch-Like ECH-Associated Protein 1/metabolism , Drosophila/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Apoptosis , Superoxide Dismutase/metabolism , Adenosine Triphosphate/pharmacologyABSTRACT
The purpose of this study was to explore the effect and mechanism of Xihuang Pills on rats with precancerous lesions of the breast. Of 48 healthy female rats, 8 were randomly selected as blank group, and the other 40 were treated with 7,12-dimethylbenzanthracene(DMBA) combined with estrogen and progestin to establish a model of precancerous lesions of the breast. The successfully modeled rats were randomly divided into a model group, a tamoxifen group(1.8 mg·kg~(-1)·d~(-1)), a Xihuang Pills low-dose group(0.3 g·kg~(-1)·d~(-1)), a medium-dose group(0.6 g·kg~(-1)·d~(-1)) and a high-dose group(1.2 g·kg~(-1)·d~(-1)). After 30 days of admi-nistration, the histopathological changes of viscera and breast were observed by haematoxylin and eosin(HE) staining, and the visceral index was calculated. Enzyme linked immunosorbent assay(ELISA) was used to detect the contents of estradiol(E_2) and progesterone(P) in serum. The protein expressions of vascular endothelial growth factor(VEGF) and fibroblast growth factor 2(FGF2) were detected by immunohistochemistry. The protein expressions of VEGF, vascular endothelial growth factor receptor 2(VEGFR2), phosphorylated-vascular endothelial growth factor receptor 2(p-VEGFR2), B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were detected by Western blot and the mRNA expressions of VEGF, FGF2, CXC-chemokine receptor 4(CXCR4), cysteine aspartic acid-specific protease(caspase-3), and stromal cell-derived factor 1(SDF-1) were detected by real-time polymerase chain reaction(RT-PCR). HE staining revealed that the model group had some liver and kidney damages and severe hyperplastic mammary tissue, while the Xihuang Pills high-dose group had mild hyperplasia. Compared with the model group, the Xihuang Pills groups had lo-wer ovarian coefficient(P<0.05 or P<0.01) and Xihuang Pills high-dose group had lower uterine coefficient(P<0.01). ELISA results showed that compared with the model group, expressions of E_2 and P in Xihuang Pills high-dose group were significantly decreased(P<0.05 or P<0.01). Immunohistochemistry, Western blot and RT-PCR indicated that compared with the conditions in the model group, the protein and mRNA expressions of VEGF and FGF2 in the Xihuang Pills groups were down-regulated(P<0.05 or P<0.01), and the protein expression of Bcl-2 was lowered(P<0.01); there was a decrease in the protein expressions of VEGFR2 and p-VEGFR2(P<0.01), a down-regulation in the mRNA expressions of CXCR4 and SDF-1(P<0.01), while an increase in the mRNA expression of caspase-3(P<0.01) in both Xihuang Pills medium-dose and high-dose groups; the protein expression of Bax in Xihuang Pills high-dose group was increased(P<0.01). The above results indicated that Xihuang Pills can effectively intervene in precance-rous lesions of the breast, and the mechanism may be related to the regulation of E_2 and P secretion as well as the inhibition of angiogenesis and chemokine receptor expression, thus controlling the occurrence of precancerous lesions of the breast in rats.
Subject(s)
Rats , Female , Animals , Rats, Sprague-Dawley , bcl-2-Associated X Protein , Vascular Endothelial Growth Factor A/metabolism , Caspase 3 , Vascular Endothelial Growth Factor Receptor-2 , Fibroblast Growth Factor 2 , Proto-Oncogene Proteins c-bcl-2 , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Precancerous Conditions , Hyperplasia , Receptors, Chemokine , RNA, MessengerABSTRACT
This study aimed to explore the anti-glioma effect of natural compound pterostilbene(PTE) through regulating pyroptosis and apoptosis pathways, and to analyze the possible anti-glioma pathways and targets of PTE by network pharmacology and molecular docking. In this study, the action targets of PTE and the glioma targets were obtained by network pharmacology to construct a target network and a protein-protein interaction(PPI) network to predict the possible action targets of PTE against glioma. Molecular docking was performed on the core targets by AutoDock and the action pathways of PTE against glioma were predicted by enrichment analysis. In addition, the effect of PTE on the viability of U87MG and GL261 glioma cells was detected by CCK-8 assay. Clone formation assay and cell scratching assay were used to explore the effect of different concentrations of PTE on the proliferation and migration, respectively of glioma cells. Hoechst staining was used to observe PTE-induced apoptosis in glioma cells. The changes in mitochondrial membrane potential were detected by JC-1 staining. The pyroptosis-inducing effect of PTE on glioma cells was observed by inverted microscopy and lactate dehydrogenase(LDH) assay. Hoechst 33342/PI dual staining assay was performed to detect the integrity of glioma cell membranes. The expressions of pyroptosis and apoptosis-related proteins in glioma cells after PTE induction were determined by Western blot. In this study, 37 anti-glioma targets of PTE were obtained, and enrichment analysis suggested that PTE exerted anti-glioma effects through various signaling pathways including cancer pathway, proteoglycan in cancer, PI3K/AKT pathway, and apoptosis regulatory pathway. Molecular docking revealed that PTE had good binding activity with the main targets. Compared with the control group, PTE significantly reduced the viability as well as the proliferation, migration and adhesion abilities of U87MG and GL261 cells; it induced the apoptosis of the two glioma cells and the decrease of mitochondrial membrane potential in U87MG cells, and the effects increased with the increase of drug concentration. Compared with the conditions in the control group, glioma cells in the PTE group had increased pyroptosis-specific appearance and gradually increased LDH release; the number of PI positive cells was significantly elevated with the increase of PTE concentration as revealed by Hoechst 33342/PI staining; the expression levels of apoptosis-related factors cleaved PARP1 and B-cell lymphoma-2(Bcl-2) associated X(BAX) in the PTE group were markedly up-regulated, while the expression level of Bcl-2 was markedly down-regulated; the activation levels of pyroptosis-related proteins cleaved caspase-3 and gasdermin E-N(GSDME-N) had a remarkable rise in the PTE group, while no significant changes were found in the activation levels of gasdermin D-N(GSDMD-N) and cleaved caspase-1. In summary, PTE plays an anti-glioma role by inhibiting cell viability, proliferation, and migration and activating the caspase-3/GSDME-mediated pyroptosis pathway and mitochondrial apoptosis pathway.
Subject(s)
Pyroptosis , Caspase 3/metabolism , Network Pharmacology , Gasdermins , Molecular Docking Simulation , Phosphatidylinositol 3-Kinases/metabolism , Apoptosis , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
OBJECTIVE@#To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML.@*METHODS@#Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR).@*RESULTS@#Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05).@*CONCLUSION@#Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.
Subject(s)
Humans , U937 Cells , Cytarabine/therapeutic use , Receptors, Interleukin-8A , NF-kappa B , Proto-Oncogene Proteins c-akt , Phosphatidylinositol 3-Kinases , Leukemia, Myeloid, Acute/genetics , Apoptosis , Cell Proliferation , Apoptosis Regulatory Proteins , Proto-Oncogene Proteins c-bcl-2 , RNA, Messenger , Cell Line, TumorABSTRACT
Objective: To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of DRAIC in lung cancer tissues and corresponding adjacent normal tissues of 40 patients with lung adenocarcinoma who underwent surgery in Tangshan People's Hospital from 2019 to 2020. Lung adenocarcinoma cells A549 and H1299 were cultured in vitro and divided into si-NC group, si-DRAIC group, miR-NC group, let-7i-5p mimics group, si-DRAIC+ inhibitor-NC group, and si-DRAIC+ let-7i-5p inhibitor group. CCK-8 method and clone formation experiment were used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Transwell array was used to detect the cell migration and invasion. Western blot was used to detect the protein expressions of Caspase-3, Caspase-9, Bcl-2 and Bax. The double luciferase reporter gene experiment was used to verify the regulatory relationship between DRAIC and let-7i-5p. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and Pearson correlation analysis was used for correlation analysis. Results: Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (P<0.05), but the expression level of let-7i-5p decreased (P<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (r=-0.737, P<0.05). The absorbance value of A549 and H1299 cells in the si-DRAIC group at 48, 72 and 96 hours were lower than those in the si-NC group (P<0.05), the number of clones formed [(91.00±6.08 vs. 136.67±6.51); (50.67±1.53 vs. 76.67±4.51)], the number of migration [(606.67±31.34 vs. 960.00±33.06); (483.33±45.96 vs. 741.67±29.67)], the number of invasion [(185.00±8.19 vs. 447.33±22.05); (365.00±33.87 vs. 688.00±32.97)] were lower than those in the si-NC group (P<0.05). However, the apoptosis rates of cells [(13.43±2.79)% vs. (4.53±0.42)%; (23.77±1.04)% vs. (6.60±1.42)%] were higher than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC group were higher than those in si-NC group, and the protein expression of Bcl-2 was lower than that in si-NC group (P<0.05). DRAIC is located in the cytoplasm. DRAIC targeted and negatively regulated the expression of let-7i-5p. The absorbance values of A549 and H1299 cells in the let-7i-5p mimics group at 48, 72 and 96 hours were lower than those in the miR-NC group (P<0.05), the number of clones formed [(131.33±14.47 vs. 171.33±6.11); (59.33±4.93 vs. 80.33±7.09)], the number of migration [(137.67±3.06 vs. 579.33±82.03); (425.00±11.14 vs. 669.33±21.13)], the number of invasion [(54.00±4.36 vs. 112.67±11.59); (80.00±4.58 vs. 333.33±16.80)] were lower than those in the miR-NC group (P<0.05). However, the apoptosis rates of cells [(14.57±1.10)% vs. (6.97±1.11)%; (23.97±0.42)% vs. (7.07±1.21)%] were higher than those in the miR-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in let-7i-5p mimics group were higher than those in miR-NC group, and the protein expression of Bcl-2 was lower than that in miR-NC group (P<0.05). The absorbance values of A549 and H1299 cells in the si-DRAIC+ let-7i-5p inhibitor group at 48, 72 and 96 hours were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05), the number of clones formed [(82.00±5.29 vs. 59.00±5.57); (77.67±4.93 vs. 41.33±7.57)], the number of migration [(774.33±35.81 vs. 455.67±19.04); (569.67±18.72 vs. 433.67±16.77)], the number of invasion [(670.33±17.21 vs. 451.00±17.52); (263.67±3.06 vs. 182.33±11.93)] were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05). However, the apoptosis rates of cells [(7.73±0.45)% vs. (19.13±1.50)%; (8.00±0.53)% vs. (28.40±0.53)%] were lower than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC+ let-7i-5p inhibitor group were higher than those in si-DRAIC+ inhibitor-NC group, and the protein expression of Bcl-2 was lower than that in si-DRAIC+ inhibitor-NC group (P<0.05). Conclusion: DRAIC is highly expressed in lung adenocarcinoma, and DRAIC promotes the proliferation, migration and invasion of lung adenocarcinoma cells and inhibits apoptosis by targeting let-7i-5p.