Mechanism of Shikonin on spinal cord injury in rats based on TNFR/RIPK1 pathway / 中国骨伤

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.

Knockdown of interferon-γ inducible protein 30 (IFI30) inhibits the proliferation, invasion and migration of human glioma U251 cells by activating STAT1 and promotes their apoptosis / 细胞与分子免疫学杂志

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.

Calcitonin gene-related peptide inhibits neuronal apoptosis in heatstroke rats via PKA/p-CREB pathway / 中华创伤杂志(英文版)

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.

Asperuloside Promotes Apoptosis of Cervical Cancer Cells through Endoplasmic Reticulum Stress-Mitochondrial Pathway / 中国结合医学杂志

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.

MiR -18a -5p aggravates homocysteine -induced myocardial injury via autophagy / 中南大学学报(医学版)

OBJECTIVES@#Hyperhomocysteinaemia (Hcy) is an independent risk factor for cardiovascular and cerebrovascular diseases. MicroRNA (miR)-18a-5p is closely related to cardiovascular diseases. This study aims to investigate the effects of miR-18a-5p on homocysteine (Hcy)-induced myocardial cells injury.@*METHODS@#H9c2 cells were transfected with miR-18a-5p mimic/miR-18a-5p mimic negative control (NC) or combined with Hcy for intervention, and untreated cells were set as a control group. The transfection efficiency was verified by real-time RT-PCR, and cell counting kit-8 (CCK-8) assay was used to determine cell viability. Flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) levels. Western blotting was performed to measure the protein levels of microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin1, p62, Bax, Bcl-2, and Notch2. Dual luciferase reporter assay was used to detect the interaction of miR-18a-5p with Notch2.@*RESULTS@#Compared with the control, treatment with Hcy or transfection with miR-18a-5p mimic alone, or combined treatment with Hcy and miR-18a-5p mimic/miR-18a-5p mimic NC significantly reduced the H9c2 cell viability, promoted apoptosis and ROS production, up-regulated the expressions of Bax and Beclin, down-regulated the expressions of Bcl-2, p62, and Notch2, and increased the ratio of LC3-II/LC3-I (all P<0.05). Compared with the combined intervention of miR-18a-5p mimic NC and Hcy group, the above indexes were more significantly changed in the combined intervention of miR-18a-5p mimic and Hcy group, and the difference between the 2 groups was statistically significant (all P<0.05). There is a targeted binding between Notch2 and miR-18a-5p.@*CONCLUSIONS@#MiR-18a-5p could induce autophagy and apoptosis via increasing ROS production in cardiomyocytes, and aggravate Hcy-induced myocardial injury. Notch2 is a target of miR-18a-5p.

AAZ2 induces mitochondrial-dependent apoptosis by targeting PDK1 in gastric cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Drastic surges in intracellular reactive oxygen species (ROS) induce cell apoptosis, while most chemotherapy drugs lead to the accumulation of ROS. Here, we constructed an organic compound, arsenical N-‍(4-(1,3,2-dithiarsinan-2-yl)phenyl)acrylamide (AAZ2), which could prompt the ROS to trigger mitochondrial-dependent apoptosis in gastric cancer (GC). Mechanistically, by targeting pyruvate dehydrogenase kinase 1 (PDK1), AAZ2 caused metabolism alteration and the imbalance of redox homeostasis, followed by the inhibition of phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway and leading to the activation of B-cell lymphoma 2 (Bcl2)/Bcl2-associated X (Bax)/caspase-9 (Cas9)/Cas3 cascades. Importantly, our in vivo data demonstrated that AAZ2 could inhibit the growth of GC xenograft. Overall, our data suggested that AAZ2 could contribute to metabolic abnormalities, leading to mitochondrial-dependent apoptosis by targeting PDK1 in GC.

Intermittent heat exposure induces thoracic aorta injury in spontaneously hypertensive rats by activating the AMPK/mTOR/ULK1 pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effects of different manners of heat exposure on thoracic aorta injury in spontaneously hypertensive rats (SHRs) and explore the underlying mechanism.@*METHODS@#Normal 6 to 7-week-old male SHRs were randomized into control group (cage at room temperature), intermittent heat exposure group (SHR-8 group, exposed to 32 ℃ for 8 h daily for 7 days) and SHR-24 group (with continuous exposure to 32 ℃ for 7 days). After the treatments, the pathologies of the thoracic aorta of the rats were observed with HE staining, and the expressions of Beclin1, LC3B and p62 were detected with Western blotting and immunofluorescence assay; TUNEL staining was used to observe cell apoptosis in the thoracic aorta, and the expressions of caspase-3, Bax, and Bcl-2 were detected using Western blotting. The effects of intraperitoneal injections of 3-MA (an autophagy agonist), rapamycin (an autophagy inhibitor) or compound C 30 min before intermittent heat exposure on the expressions of proteins associated with autophagy, apoptosis and the AMPK/mTOR/ULK1 pathway in the aorta were examined with immunohistochemistry.@*RESULTS@#In SHR-8 group, the rats showed incomplete aortic intima with disordered cell distribution and significantly increased expressions of Beclin1, LC3II/LC3I and Bax, lowered expressions of p62 and Bcl-2, and increased apoptotic cells in the thoracic aorta (P < 0.05). Pretreatment with 3-MA obviously inhibited the expressions of autophagy- and apoptosis-related proteins, whereas rapamycin promoted their expressions. Compared with the control group, the rats in SHR-8 group had significantly down-regulated p-mTOR and up-regulated p-AMPK and p-ULK1 expression of in the aorta; Treatment with compound C obviously lowered the expressions of p-AMPK and p-ULK1 and those of LC3B and Beclin1 as well.@*CONCLUSION@#In SHRs, intermittent heat exposure causes significant pathologies and promotes autophagy and apoptosis in the thoracic aorta possibly by activating the AMPK/mTOR/ULK1 pathway.

Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway / 中国中药杂志

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.

Stellera chamaejasme extract against multidrug resistance of breast cancer cell line MCF-7 / 中国中药杂志

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.

Alcohol extract of root and root bark of Toddalia asiatica alleviates CIA in rats through anti-inflammatory and proapoptotic effects / 中国中药杂志

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.

Neuroprotective effect of ginsenoside Re on drosophila model of Parkinson's disease / 中国中药杂志

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.

Effect and mechanism of Xihuang Pills on rats with precancerous lesions of breast / 中国中药杂志

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.

Anti-glioma mechanism of pterostilbene by regulating apoptosis and GSDME-mediated pyroptosis pathways: a study based on network pharmacology and experimental research / 中国中药杂志

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.

Effects of Xihuang Pills on proliferation and apoptosis of prostate cancer LNCaP cells based on AR/m TOR signaling pathway / 中国中药杂志

Based on the androgen receptor(AR)/mammalian target of rapamycin(mTOR)signaling pathway, the effects of Xihuang Pills-medicated serum on the proliferation and apoptosis of prostate cancer LNCaP cells were investigated. The drug-containing serum of SD rats was prepared by intragastric administration of Xihuang Pills suspension. The effects of low-, medium-, and high-dose Xihuang Pills-containing serum on the in vitro proliferation of LNCaP cells were detected by cell counting kit-8(CCK-8). Flow cytometry was used to detect the apoptosis level of LNCaP cells after intervention with different concentrations of Xihuang Pills. Protein expression of cleaved cysteinyl aspartate-specific proteinase caspase-3(cleaved caspase-3), B-cell lymphoma-2(Bcl-2), and AR as well as the phosphorylation level of mTOR protein were detected by Western blot. The results showed that compared with the blank serum, the drug-medicated serum could blunt the activity of LNCaP cells. Low-, medium-, and high-dose Xihuang Pills-containing serum could significantly increase the cell apoptosis rate, increase the expression of cleaved caspase-3 protein, decrease the expression of Bcl-2 protein, reduce the expression of AR protein, and down-regulate the level of phosphorylated mTOR(p-mTOR). To study the effect of Xihuang Pills on the growth of LNCaP cells in vivo, different doses of Xihuang Pills were used to intervene in the subcutaneous graft model in nude mice inoculated with LNCaP cells. The expression levels of AR, mTOR, p-mTOR, Bcl-2, and cleaved caspase-3 were detected by Western blot. The results showed that the volumes of subcutaneous graft tumor in the low-dose, medium-dose, and high-dose Xihuang Pills groups significantly decreased compared with that in the model group. The weight of subcutaneous transplanted tumor in each group with drug intervention was significantly lower than that in the model group. Compared with the model group, the low-dose, medium-dose, and high-dose Xihuang Pills groups showed increased cleaved caspase-3 protein expression, decreased Bcl-2 and AR protein expression, and reduced p-mTOR protein expression. Further experiments showed that AR agonist R1881 could block the anti-proliferation and pro-apoptotic effects of Xihuang Pills. The mechanism of Xihuang Pills against prostate cancer is related to the inhibition of the AR/mTOR signaling pathway, inhibition of LNCaP cell proliferation, and induction of apoptosis in cancer cells.

Qirong Tablets inhibits apoptosis of ovarian granulosa cells via PI3K/Akt/ HIF-1 signaling pathway / 中国中药杂志

This study aims to observe the effect and explore the mechanism of Qirong Tablets in the treatment of premature ovarian insufficiency(POI) in mice via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor 1(HIF-1) signaling pathway. Sixty SPF female BALB/c mice were randomly divided into normal group, model group, positive control group, Qirong Tablets low-, medium-and high-dose group. The normal group was intraperitoneally injected with the same amount of normal saline, and the other groups were intraperitoneally injected with cyclophosphamide 120 mg·kg~(-1)·d~(-1) once to establish a POI animal model. After the model was successfully established, the low-, medium-and high-dose groups of Qirong Tablets were administered orally with 0.6, 1.2, 2.4 mg·kg~(-1)·d~(-1) respectively. The positive control group was given 0.22 mg·kg~(-1)·d~(-1) Clementine Tablets by intragastric administration, and the normal group and model group were given intragastric administration with the same amount of normal saline, and the treatment was 28 d as a course of treatment. After drug intervention, enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of estradiol(E_2), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and anti-mullerian hormone(AMH) in peripheral blood, and hematoxylin-eosin(HE) staining to observe the ovarian tissue. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to detect the apoptosis of granulosa cells, and Western blot to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, PI3K, Akt, and HIF-1. Compared with the normal group, the modeling of POI caused loose or destroyed ovarian tissue with vacuolar structures, edema and fibrosis in the ovarian interstitium, disordered or loose arrangement of granulosa cells, and reduced normal follicles. Compared with the model group, drug interventions restored the ovarian tissue and follicles at all the development stages and reduced atretic follicles. Compared with the normal group, the modeling of POI lowered the serum level of E_2 and AMH(P<0.01), and elevated the level of FSH and LH(P<0.01). Compared with the model group, high-dose Qirong Tablets elevated the levels of E_2 and AMH(P<0.05), and lowered the levels of FSH and LH(P<0.05). Compared with the normal group, the modeling of POI up-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 and down-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.01). Compared with the model group, low-, medium-, and high-dose Qirong Tablets down-regulated the protein levels of PI3K, Akt, HIF-1, Bax, and caspase-3 proteins and up-regulated the protein level of Bcl-2 in the ovarian tissue(P<0.05). In conclusion, Qirong Tablets can up-regulate the expression Bcl-2, down-regulate the expression of Bax and caspase-3 in POI mice. Qirong Tablets may inhibit the apoptosis of follicular granulosa cells in mice, thereby delaying ovarian aging, improving reproductive axis function, and strengthening ovarian reserve capacity, which may be associated with the inhibition of PI3K/Akt/HIF-1 pathway.

Blaps rynchopetera combined with cyclophosphamide affects proliferation and apoptosis of lung cancer cells via Wnt/β-catenin signaling pathway / 中国中药杂志

This study aims to investigate the effects of Blaps rynchopetera Fairmaire and/or cyclophosphamide on the proliferation and apoptosis of lung cancer cells and decipher the underlying mechanism. B. rynchopetera and cyclophosphamide-containing serum and blank serum were prepared from SD rats. Cell counting kit-8(CCK-8) assay was employed to examine the proliferation of lung cancer cell lines A549 and Lewis treated with corresponding agents. The Jin's formula method was used to evaluate the combined effect of the two drugs. According to the evaluation results, appropriate drug concentrations and lung cancer cell line were selected for subsequent experiments, which included control, B. rynchopetera, cyclophosphamide, B. rynchopetera + cyclophosphamide, and B. rynchopetera + Wnt/β-catenin pathway agonist lithium chloride(LiCl) groups. Immunocytochemistry was employed to measure the expression of proliferation-related proteins in Lewis cells after drug interventions. Flow cytometry was employed to determine the cell cycle and apoptosis. The expression levels of proliferating cell nuclear antigen(PCNA), cyclinD1, B-cell lymphoma 2(Bcl-2), Bcl-2-assiocated X protein(Bax), Wnt1, and β-catenin were determined by Western blot. The results showed that B. rynchopetera and/or cyclophosphamide significantly inhibited the proliferation of A549 and Lewis cells. Compared with B. rynchopetera alone, the combination increased the inhibition rate on cell proliferation. The combination of B. rynchopetera and cyclophosphamide demonstrated a synergistic effect according to Jin's formula-based evaluation. Compared with the control group, the B. rynchopetera, cyclophosphamide, and B. rynchopetera + cyclophosphamide groups showed increased proportion of Lewis cells in G_0/G_1 phase, increased apoptosis rate, up-regulated expression of Bax, and down-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and β-catenin. Compared with the cyclophosphamide group, the combination group showed increased proportion of cells in G_0/G_1 phase, increased apoptosis rate, up-regulated expression of Bax, and down-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and β-catenin. Compared with the B. rynchopetera group, the B. rynchopetera + LiCl group had deceased proportion of cells in G_0/G_1 phase, decreased apoptosis rate, down-regulated expression of Bax, and up-regulated expression of PCNA, cyclinD1, Bcl-2, Wnt1, and β-catenin. The results indicated that B. rynchopetera could inhibit the proliferation, arrest the cell cycle, and induce the apoptosis of lung cancer cells by inhibiting the Wnt/β-catenin signaling pathway. Moreover, B. rynchopetera had a synergistic effect with cyclophosphamide.

Effect and mechanism of Danggui Buxue Decoction-containing serum in mitigating H9c2 cell injury caused by exposure to intermittent low oxygen / 中国中药杂志

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.

Mechanism of protective effect of resveratrol on poor ovarian response in mice / 中国中药杂志

This study aims to investigate the therapeutic effects and potential mechanisms of resveratrol(Res) on poor ovarian response(POR) in mice. The common target genes shared by Res and POR were predicted by network pharmacology, used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and then validated by animal experiments. The mice with regular estrous cycle after screening were randomized into normal, POR, and low-and high-dose(20 and 40 mg·kg~(-1), respectively) Res groups. The normal group was administrated with an equal volume of 0.9% sodium chloride solution by gavage, and the mice in other groups with tripterygium glycosides suspension(50 mg·kg~(-1)) by gavage for 2 weeks. After the modeling, the mice in low-and high-dose Res groups were treated with Res by gavage for 2 weeks, and the mice in normal and POR groups with an equal volume of 0.9% sodium chloride solution by gavage. Ovulation induction and sample collection were carried out on the day following the end of treatment. Vaginal smears were collected for observation of the changes in the estrous cycle, the counting of retrieved oocytes, and the measurement of ovarian wet weight and ovarian index. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of anti-mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in the serum. The ovarian tissue morphology and granulosa cell apoptosis were observed by hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL), respectively. Western blot was employed to determine the protein levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(AKT), forkhead box O(FOXO) 3a, hypoxia-inducible factor(HIF)-1α, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). A total of 222 common targets shared by Res and POR were collected. GO annotation indicated that these targets were mainly involved in oxidative stress response. KEGG enrichment analysis revealed that Res can intervene in POR via PI3K/AKT, HIF-1, and FOXO signaling pathways. Animal experiments showed that the model group had higher rate of estrous cycle disorders, lower number and poorer morphology of normally developed follicles at all levels, more atretic follicles, higher apoptosis of ovarian granulosa cells, lower number of retrieved oocytes, lower ovarian wet weight and ovarian index, higher serum levels of FSH and LH, lower levels of AMH and E_2, higher expression levels of HIF-1α, FOXO3a and Bax, and lower expression levels of PI3K, AKT, and Bcl-2 in the ovarian tissue than the normal group. Compared with the POR group, low-and high-dose Res decreased the rate of estrous cycle disorders, improved the follicle number and morphology, reduced atretic follicles, promoted the apoptosis of ovarian granulosa cells, increased retrieved oocytes, ovarian wet weight and ovarian index, and lowered serum FSH and LH levels. Moreover, Res down-regulated the expression levels of HIF-1α, FOXO3a and Bax, and up-regulated the expression levels of PI3K, AKT and Bcl-2 in the ovarian tissue. In summary, Res can inhibit apoptosis and mitigate poor ovarian response in mice by regulating the PI3K/AKT/FOXO3a and HIF-1α pathways.

Effect and mechanism of Poria cocos polysaccharides on myocardial cell apoptosis in rats with myocardial ischemia-reperfusion injury by regulating Rho-ROCK signaling pathway / 中国中药杂志

This study aimed to investigate the effect and underlying mechanism of Poria cocos polysaccharides(PCP) on myocardial cell apoptosis in the rat model of myocardial ischemia-reperfusion injury(MI/RI). Male SPF-grade SD rats were randomly divided into a sham group(saline), a model group(saline), low-and high-dose PCP groups(100 and 200 mg·kg~(-1)), and a fasudil group(10 mg·kg~(-1)), with 16 rats in each group. Except for the sham group, the other four groups underwent left anterior descending coronary artery ligation for 30 min followed by reperfusion for 2 h to establish the MI/RI model. The myocardial infarct area was assessed by TTC staining. Histological changes were observed through HE staining. Myocardial cell apoptosis was evaluated using TUNEL staining. Serum lactate dehydrogenase(LDH), creatine kinase MB(CK-MB), interleukin-1β(IL-1β) and IL-18 levels, myocardial superoxide dismutase(SOD) activity and malondialdehyde(MDA) levels were detected by ELISA. Protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved caspase-3, Ras homolog gene A(RhoA), myosin phosphatase target subunit 1(MYPT-1), phosphorylated MYPT-1(p-MYPT-1), and Rho-associated coiled-coil forming kinase 1(ROCK 1) were measured by Western blot. Pathological staining of myocardial tissue revealed that in the model group, there was focal necrosis of myocardial tissue, myocardial cell swelling, unclear boundaries, and neutrophil infiltration. These pathological changes were alleviated in the low-and high-dose PCP groups and the fasudil group. Compared with the model group, the low-and high-dose PCP groups and the fasudil group showed significantly reduced myocardial infarct area and myocardial cell apoptosis rate. Compared with the sham group, the model group exhibited elevated serum LDH, CK-MB, IL-1β and IL-18 levels, increased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and decreased myocardial SOD levels and Bcl-2 protein expression. Compared with the model group, the PCP groups and the fasudil group showed lowered serum LDH, CK-MB, IL-1β and IL-18 levels, decreased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and increased myocardial SOD levels and Bcl-2 protein expression. PCP exhibited a certain preventive effect on myocardial tissue pathological damage and myocardial cell apoptosis in MI/RI rats, possibly related to the inhibition of the Rho-ROCK signaling pathway activation, thereby reducing oxidative stress and inflammatory responses.

LncRNA DRAIC regulates the proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells by targeting let-7i-5p / 中华肿瘤杂志

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.