Lipid unsaturation promotes BAX and BAK pore activity during apoptosis.

BAX and BAK are proapoptotic members of the BCL2 family that directly mediate mitochondrial outer membrane permeabilition (MOMP), a central step in apoptosis execution. However, the molecular architecture of the mitochondrial apoptotic pore remains a key open question and especially little is known about the contribution of lipids to MOMP. By performing a comparative lipidomics analysis of the proximal membrane environment of BAK isolated in lipid nanodiscs, we find a significant enrichment of unsaturated species nearby BAK and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids promote BAX pore activity in model membranes, isolated mitochondria and cellular systems, which is further supported by molecular dynamics simulations. Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA release. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides an insight on how local lipid environment affects BAX and BAK function during apoptosis.

Apoptotic effect of 5-fluorouracil-doxorubicin combination on colorectal cancer cell monolayers and spheroids.

BACKGROUND: Drug combination studies help to improve new treatment approaches for colon cancer. Tumor spheroids (3D) are better models than traditional 2-dimensional cultures (2D) to evaluate cellular responses to chemotherapy drugs. The cultivation of cancer cells in 2D and 3D cultures affects the apoptotic process, which is a major factor influencing the response of cancer cells to chemotherapeutic drugs. In this study, the antiproliferative effects of 5-fluorouracil (5-FU) and doxorubicin (DOX) were investigated separately and in combination using 2D and 3D cell culture models on two different colon cancer cell lines, HT-29 (apoptosis-resistant cells) and Caco-2 2 (apoptosis-susceptible cells). METHODS: The effect of the drugs on the proliferation of both colon cancer cells was determined by performing an MTT assay in 2D culture. The apoptotic effect of 5-FU and DOX, both as single agents and in combination, was assessed in 2D and 3D cultures through quantitative real-time polymerase chain reaction analysis. The expression of apoptotic genes, such as caspases, p53, Bax, and Bcl-2, was quantified. RESULTS: It was found that the mRNA expression of proapoptotic genes was significantly upregulated, whereas the mRNA expression of the antiapoptotic Bcl-2 gene was significantly downregulated in both colon cancer models treated with 5-FU, DOX, and 5-FU + DOX. CONCLUSION: The results indicated that the 5-FU + DOX combination therapy induces apoptosis and renders 5-FU and DOX more effective at lower concentrations compared to their alone use. This study reveals promising results in reducing the potential side effects of treatment by enabling the use of lower drug doses.

Vitamin B6 ameliorates acute pancreatitis by suppressing the caspase3 signaling pathway.

BACKGROUND: Acute pancreatitis (AP) is a prevalent exocrine inflammatory disorder of the pancreas characterized by pancreatic inflammation and injury to acinar cells. Vitamin B6 (VB6) is a vital nutrient that plays a significant role in preserving human health and has anti-inflammatory and anti-apoptotic effects. METHODS: This study aimed to explore the potential pancreatic protective effects of VB6 in mitigating pancreatic inflammation and apoptosis induced by taurocholate sodium (TLCS) in an AP model and to assess the underlying mechanism of action. AP was induced in Sprague‒Dawley (SD) rats through TLCS administration and lipopolysaccharide (LPS)-treated AR42J cells, followed by treatment with VB6. RESULTS: Various parameters associated with AP were assessed in both plasma and pancreatic tissues. VB6 has been shown to ameliorate the severity of AP through various mechanisms. It effectively reduces the levels of serum amylase, lipase, and inflammatory factors, thereby mitigating histological injury to the pancreas. Moreover, VB6 inhibited pancreatic apoptosis by downregulating bax expression and up-regulating Bcl2 expression in TLCS-treated rats. Additionally, VB6 suppressed the expression of caspase3. The anti-inflammatory and anti-apoptotic effects of VB6 observed in LPS-treated AR42J cells are consistent with those observed in a rat model of AP. CONCLUSIONS: These results suggest that VB6 exerts anti-inflammatory and anti-apoptotic effects through inhibition of the caspase3 signaling pathway and has a protective effect against AP.

Dermaseptin B2 bioactive gene's potential for anticancer and anti-proliferative effect is linked to the regulation of the BAX/BBC3/AKT pathway.

Dermaseptin B2 (DrsB2) is an antimicrobial peptide with anticancer and angiostatic properties. We aimed to assess the in vitro inhibitory effect of pDNA/DrsB2 on the growth of breast cancer cells and its impact on the expression of genes involved in the BAX/BBC3/AKT pathway. The nucleic acid sequence of DrsB2 was artificially synthesized and inserted into the pcDNA3.1( +) Mammalian Expression Plasmid. PCR testing and enzyme digesting procedures evaluated the accuracy of cloning. The vectors were introduced into cells using LipofectamineTM2000 transfection reagent. The breast cancer cells were assessed by flow cytometry, MTT assessment, soft agar colony method, and wound healing investigation. The gene's transcription was evaluated using real-time PCR with a significance level of P < 0.05. The recombinant plasmid harboring the pDNA/DrsB2 vector was effectively produced, and the gene sequence showed absolute homogeneity (100% similarity) with the DrsB2 gene. The transfection effectiveness of MCF-7 and MCF-10A cells was 79% and 68%, respectively. The findings are measured using the growth inhibition 50% (GI50) metric, which indicates the concentration of pDNA/DrsB2 that stops 50% of cell growth. The proportions of early apoptosis, late apoptosis, necrosis, and viable MCF-7 cells in the pDNA/DrsB2 group were 40.50%, 2.31%, 1.69%, and 55.50%, respectively. The results showed a 100% increase in gene expression in programmed cell death following treatment with pDNA/DrsB2 (**P < 0.01). To summarize, the results described in this work offer new possibilities for treating cancer by targeting malignancies via pDNA/DrsB2 and activating the BAX/BBC3/AKT signaling pathways.

Ketotifen counteracts cisplatin-induced acute kidney injury in mice via targeting NF-κB/NLRP3/Caspase-1 and Bax/Bcl2/Caspase-3 signaling pathways.

Cisplatin (CIS) stands as one of the most effective chemotherapy drugs currently available. Despite its anticancer properties, the clinical application of CIS is restricted due to nephrotoxicity. Our research aimed to specify the impact of ketotifen fumarate (KET) against nephrotoxicity induced by CIS in mice. Male NMRI mice were treated with KET (0.4, 0.8, and 1.6 mg/kg, ip) for seven days. On the fourth day of the study, a single dose of CIS (13 mg/kg, ip) was administered, and the mice were sacrificed on the eighth day. The results indicated that administration of KET attenuated CIS-induced elevation of BUN and Cr in the serum, as well as renal KIM-1 levels. This improvement was accompanied by a significant reduction in kidney tissue damage, which was supported by histopathological examinations. Likewise, the decrease in the ratio of GSH to GSSG and antioxidant enzyme activities (CAT, SOD, and GPx), and the increase in lipid peroxidation marker (TBARS) were reversed in KET-treated mice. The ELISA results revealed that KET-treated mice ameliorated CIS-induced elevation in the renal levels of TNF-α, IL-1ß, and IL-18. Western blot analysis exhibited that KET suppressed the activation of the transcription factor NF-κB and the NLRP3 inflammasome in the kidney of CIS-treated mice. Moreover, KET treatment reversed the changes in the protein expression of markers related to apoptosis (Bax, Bcl2, Caspase-3, and p53). Interestingly, KET significantly enhanced the cytotoxicity of CIS in HeLa cells. In conclusion, this study provides valuable insights into the promising effects of KET in mitigating CIS-induced nephrotoxicity.

Ptehosides A-I: Nine undescribed iridoids with in vitro cytotoxicity from the whole plant of Pterocephalus hookeri (C.B. Clarke) Höeck.

Nine previously undescribed iridoids, ptehosides A-I (1-9), together with 12 known ones (10-21), were isolated from Pterocephalus hookeri (C.B. Clarke) Höeck. Their structures were elucidated using various spectroscopic methods including HR-ESI-MS, NMR, UV, IR and CD, etc. The cytotoxic activities of all isolates were evaluated using MTT method in three human cancer cell lines (Caco2, Huh-7, and SW982). As result, compound 9 exhibited substantial inhibitory activity on Caco2, Huh-7, and SW982 cells with IC50 values of 1.17 ± 0.05, 1.15 ± 0.05 and 1.14 ± 0.04 µM, respectively. A preliminary mechanism study showed that 9 arrested the cell cycle of SW982 cells in the G0/G1 phase and induced apoptosis by upregulating Bax expression and downregulating Bcl-2 expression.

Evaluation of the cytotoxic activities of the essential oil from Pistacia atlantica Desf. oleoresin on human gastric cancer cell lines.

Numerous herbal products have been the subject of research regarding their potential role in cancer prevention or adjuvant therapy. Pistacia atlantica and its main phytochemicals have garnered significant attention for their potential anti-cancer effects. The study aimed to assess the growth inhibitory effects of P. atlantica essential oil (PAEO) on MKN-45 and AGS cells. This study quantified the volatile compounds in PAEO using Gas Chromatography-Mass Spectrometry (GC-MS). Subsequently, MKN-45 and AGS cells were treated with varying concentrations of PAEO (5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.156%, 0.0781%, 0.0391%, 0.0195%) for 24 h. Cell viability was evaluated through the MTT assay. The impact of PAEO on gene expression was investigated by quantifying the mRNA levels of Bax and Bcl2 in the various experimental groups using quantitative Real-Time PCR (qRT-PCR) analysis. Additionally, flow cytometry was utilized to evaluate apoptosis in the treated cells. The analysis of PAEO revealed that α-pinene was the predominant monoterpene, constituting 87.9% of the oil composition. The cytotoxic effects of PAEO were evaluated, and it was found that the oil significantly reduced the viability of MKN-45 and AGS cells. The IC50 for MKN-45 cells was determined to be 1.94 × 10-3% after 24 h of treatment, while for AGS cells the IC50 was 2.8 × 10-3% after 24 h. Additionally, the research revealed that PAEO triggered a notable rise in apoptotic cells in both AGS and MKN-45 cell lines. Moreover, at the molecular level, the findings indicated an increase in Bax expression and a decrease in Bcl2 mRNA expression, providing further evidence of the induction of apoptosis in both MKN-45 and AGS cell lines following PAEO treatment. The findings of this study offer evidence supporting the cytotoxic effects of PAEO on gastric cancer cell lines by promoting apoptosis. The findings suggest that PAEO may offer potential as a therapeutic candidate in managing and treating gastric cancer.

Trichinella spiralis Larval Extract as a Biological Anti-Tumor Therapy in a Murine Model of Ehrlich Solid Carcinoma.

Trichinella spiralis (T. spiralis) is an immunomodulating parasite that can adversely affect tumor growth and extend host lifespan. The aim of this study was to elucidate the mechanisms by which T. spiralis larval antigens achieve this effect using Ehrlich solid carcinoma (ESC) murine model. Assessment was done by histopathological and immunohistochemical analysis of caspase-3, TNF-α, Ki-67 and CD31. Additionally, Bcl2 and Bcl2-associated protein X (Bax) relative gene expression was assessed by molecular analysis for studying the effect of T. spiralis crude larval extract (CLE) antigen on tumor necrosis, apoptosis, cell proliferation and angiogenesis. We found that both T. spiralis infection and CLE caused a decrease in the areas of necrosis in ESC. Moreover, they led to increased apoptosis through activation of caspase-3, up-regulation of pro-apoptotic gene, Bax and down-regulation of anti-apoptotic gene, Bcl2. Also, T. spiralis infection and CLE diminished ESC proliferation, as evidenced by decreasing Ki-67. T. spiralis infection and CLE were able to suppress the development of ESC by inhibiting tumor proliferation, inducing apoptosis and decreasing tumor necrosis, with subsequent decrease in tumor metastasis. T. spiralis CLE antigen may be considered as a promising complementary immunotherapeutic agent in the treatment of cancer.

Rotator Cuff Tendinopathy: Pathways of Apoptosis.

Rotator cuff repair is usually successful, but retear is not uncommon. It has been previously identified that there is a higher incidence of apoptosis in the edges of the torn supraspinatus tendon. A prospective cohort study was conducted with 28 patients-14 rotator cuff tear patients, 5 instability patients, and 9 Anterior cruciate ligament reconstruction patients to determine whether there was any increase in several genes implicated in apoptosis, including Fas receptor (FasR), Fas ligand, Aifm-1, Bcl-2, Fadd, Bax, and caspase-3. There was a significant expression of Bax (P=0.2) and FasR (P=0.005) in the edges of torn supraspinatus tendons, and in intact subscapularis tendons, there was a significant expression of caspase-3 (P=0.02) compared with samples from the torn supraspinatus tendon (P=0.04). The cytochrome c pathway, with its subsequent activation of caspase-3, as well as the TRAIL-receptor signaling pathway involving FasR have both been implicated. The elevated expression of Bax supported the model that the Bax to Bcl-2 expression ratio represents a cell death switch. The elevated expression of Bax in the intact subscapularis tissue from rotator cuff tear patients also may confirm that tendinopathy is an ongoing molecular process.

Platelet microparticles influence gene expression and modulate biological activities of chronic myeloid leukemia cells (K562).

BACKGROUND: The current understanding emphasizes the intricate interplay between the Leukemic cell and its environment. Platelet-derived microparticles play a crucial role in facilitating intercellular communication and contribute to the complex landscape of cancer pathology. This study aimed to investigate the influence of platelet-derived microparticles on cell proliferation, apoptosis, and the expression of key genes, including P53, P21, Cyclin D1, Bax, and Bcl-2, within the context of a chronic myeloid leukemia cell line (K562). METHODS AND RESULTS: Platelet-derived microparticles were obtained through centrifugation at various speeds, and their concentration was quantified using the BCA assay. To determine the size and immunophenotypic characteristics of the PMPs, both the DLS technique and flow cytometry were employed. Cell proliferation was assessed using the MTT assay and hemocytometer, and cell cycle analysis was conducted through DNA content evaluation. Real-time PCR was utilized for gene expression analysis of Bax, Bcl-2, Cyclin D1, P53, and P21. Flow cytometry was employed to examine cell apoptosis. The findings revealed that platelet-derived microparticles have the ability to decrease proliferation of the K562 cell line, while not exerting an impact on apoptosis and cell cycle progression. Analysis through real-time PCR indicated an upregulation in the gene expression of P53, P21, and Bcl-2, accompanied by a downregulation in Bax and Cyclin D1. CONCLUSION: This investigation sheds light on the intricate relationship between chronic myeloid leukemia and its microenvironment, particularly the involvement of platelet-derived microparticles. The study underscores the potential of platelet-derived microparticles to influence cell behavior and gene expression, providing a deeper understanding of their role in CML and its therapeutic implications.

FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury.

BACKGROUND: Neutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury (TBI). However, the heterogeneity, multifunctionality, and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood. METHODS: Using the combined single-cell transcriptomics, metabolomics, and proteomics analysis from TBI patients and the TBI mouse model, we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests. We also characterized the underlying mechanisms both in vitro and in vivo through molecular simulations, signaling detections, gene expression regulation assessments [including dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays], primary cultures or co-cultures of neutrophils and oligodendrocytes, intracellular iron, and lipid hydroperoxide concentration measurements, as well as forkhead box protein O1 (FOXO1) conditional knockout mice. RESULTS: We identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model. Infiltration of these FOXO1high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI, aggravating acute brain inflammatory damage and promoting late TBI-induced depression. In the acute stage, FOXO1 upregulated cytoplasmic Versican (VCAN) to interact with the apoptosis regulator B-cell lymphoma-2 (BCL-2)-associated X protein (BAX), suppressing the mitochondrial translocation of BAX, which mediated the antiapoptotic effect companied with enhancing interleukin-6 (IL-6) production of FOXO1high neutrophils. In the chronic stage, the "FOXO1-transferrin receptor (TFRC)" mechanism contributes to FOXO1high neutrophil ferroptosis, disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein, which contributes to the progression of late depression after TBI. CONCLUSIONS: FOXO1high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI, which provides insight into the heterogeneity, reprogramming activity, and versatility of neutrophils in TBI.

Evaluation of Some Prognostic Biomarkers in Human Papillomavirus-Related Multiphenotypic Sinonasal Carcinoma.

Background: Human papillomavirus (HPV)-related multi phenotypic sinonasal carcinoma (HMSC) is a recently described tumor subtype with an unknown prognosis, often misdiagnosed with other sinonasal carcinomas, and associated with high-risk HPV (HR-HPV). The present study aimed to evaluate the expression of vascular endothelial growth factor (VEGF), Bcl-2-associated X protein (BAX), epidermal growth factor receptors (EGFR), ProExTMC, and human telomerase reverse transcriptase (hTERT) and assess their association with survival and clinicopathological characteristics. Methods: Between 2017 and 2022, 40 HMSC patients underwent surgical resection at the School of Medicine, Zagazig University Hospitals (Zagazig, Egypt). Tissue samples were examined for the presence of HR-HPV; absence of myeloblastosis (MYB), MYB proto-oncogene like 1 (MYBL1), and nuclear factor I/B (NFIB) fusions and the presence of myoepithelial proteins (calponin, S100, SMA), squamous differentiation markers (p63, p40, calponin), VEGF, BAX, ProExTMC, and hTERT by immunohistochemistry. All patients were followed up for about 54 months until death or the last known survival data. Data were analyzed using the Chi square test and Kaplan-Meier method. Results: The expression of VEGF, hTERT, and ProExTMC was significantly associated with age, advanced tumor stages, lymph node metastasis, tumor size, mortality, relapse, poor disease-free survival (DFS), and overall survival (OS) (P<0.001). BAX expression was significantly associated with tumor size, age, poor DFS, and relapse (P=0.01, P<0.001, P=0.035, and P=0.002, respectively). Conclusion: HMSC is strongly associated with HR-HPV. The expression of VEGF, EGFR, BAX, hTERT, and ProExTMC is associated with aggressive malignant behavior, poor survival, and poor prognosis, making them novel prognostic biomarkers for targeted therapeutics in HMSC.

[HTD4010 attenuates myocardial injury in mice with septic cardiomyopathy by promoting autophagy via the AMPK/mTOR signaling pathway].

OBJECTIVE: To investigate the protective effects of HTD4010 against lipopolysaccharide (LPS)-induced septic cardiomyopathy (SCM) in mice and explore the mechanisms mediating its effect. METHODS: Forty-five male ICR mice were randomized equally into control group, LPS (10 mg/kg) group, and LPS+HTD4010 group (in which 2.5 mg/kg HTD4010 was injected subcutaneously at 1 h and 6 h after LPS injection). Cardiac function of the mice was evaluated by ultrasound, and pathological changes in the myocardial tissues were observed with HE staining. The levels of IL-6 and TNF-α in serum and myocardial tissues were detected using ELISA, and apoptosis of the cardiomyocytes was detected with TUNEL staining. The expression levels of the key proteins associated with apoptosis, autophagy and the AMPK/mTOR pathway in the myocardial tissues were detected using Western blotting. The ultrastructural changes of cardiac myocardial mitochondria was observed with transmission electron microscopy. RESULTS: LPS exposure caused severe myocardial damage in mice, characterized by myocardial fiber rupture, structural disorder, inflammatory cell infiltration, and mitochondrial damage. The LPS-treated mice exhibited significantly decreased cardiac LVEF and FS values, elevated IL-6 and TNF-αlevels in serum and myocardial tissue, and an increased myocardial cell apoptosis rate with enhanced expressions of Bax, p-62 and p-mTOR and lowered expressions of Bcl-2, LC3 II/I, Beclin-1 and p-AMPK (P < 0.05 or 0.01). Treatment of the septic mice with HTD4010 significantly alleviated myocardial damage, increased LVEF and FS values, reduced IL-6 and TNF-α levels in serum and myocardial tissue, decreased cardiomyocyte apoptosis, lowered myocardial expressions of Bax, p-62 and p-mTOR, and increased Bcl-2, LC3 II/I, Beclin-1 and p-AMPK expressions (P < 0.05 or 0.01). CONCLUSION: HTD4010 can attenuate myocardial injury in SCM mice possibly by promoting autophagy via modulating the AMPK/mTOR signaling pathway.

[Mechanism of resveratrol in protecting ovary in poor ovarian response mice by regulating Hippo signaling pathway].

This study observed the protective effect of resveratrol(Res) on ovarian function in poor ovarian response(POR) mice by regulating the Hippo signaling pathway and explored the potential mechanism of Res in inhibiting ovarian cell apoptosis. Female mice with regular estrous cycles were randomly divided into a blank group, a model group, and low-and high-dose Res groups(20 and 40 mg·kg~(-1)), with 20 mice in each group. The blank group received an equal volume of 0.9% saline solution by gavage, while the model group and Res groups received suspension of glycosides of Triptergium wilfordii(GTW) at 50 mg·kg~(-1) by gavage for two weeks to induce the model. After modeling, the low-and high-dose Res groups were continuously treated with drugs by gavage for two weeks, while the blank group and the model group received an equal volume of 0.9% saline solution by gavage. Ovulation was induced in all groups on the day following the end of treatment. Finally, 12 female mice were randomly selected from each group, and the remaining eight female mice were co-housed with male mice at a ratio of 1∶1. Changes in the estrous cycle of mice were observed using vaginal cytology smears. The number of ovulated eggs, ovarian wet weight, ovarian index, and pregnancy rate of mice were measured. The le-vels of anti-Mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in serum were determined using enzyme-linked immunosorbent assay(ELISA). Ovarian tissue morphology and ovarian cell apoptosis were observed using hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining, respectively. The protein expression levels of yes-associated protein(YAP) 1 and transcriptional coactivator with PDZ-binding motif(TAZ) were detected by immunohistochemistry(IHC), while the changes in protein expression levels of mammalian sterile 20-like kinase(MST) 1/2, large tumor suppressor(LATS) 1/2, YAP1, TAZ, B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were determined by Western blot. The results showed that compared with the blank group, the model group had an increased rate of estrous cycle disruption in mice, a decreased number of normally developing ovarian follicles, an increased number of blocked ovarian follicles, increased ovarian granulosa cell apoptosis, decreased ovulation, reduced ovarian wet weight and ovarian index, increased serum FSH and LH levels, decreased AMH and E_2 levels, decreased protein expression levels of YAP1 and TAZ in ovarian tissues, increased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and decreased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Additionally, the number of embryos per litter significantly decreased after co-housing. Compared with the model group, the low-and high-dose Res groups exhibited reduced estrous cycle disruption rates in mice, varying degrees of improvement in the number and morphology of ovarian follicles, reduced numbers of blocked ovarian follicles, improved ovarian granulosa cell apoptosis, increased ovulation, elevated ovarian wet weight and ovarian index, decreased serum FSH and LH levels, increased AMH and E_2 levels, elevated protein expression levels of YAP1 and TAZ in ovarian tissues, decreased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and increased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Furthermore, the number of embryos per litter increased to varying degrees after co-housing. In conclusion, Res effectively inhibits ovarian cell apoptosis in mice and improves ovarian responsiveness. Its mechanism may be related to the regulation of key molecules in the Hippo pathway.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

[Bupleuri Radix-Paeoniae Radix Alba medicated plasma exerts effects on HepG2 hepatoma cells by regulating miR-1297/PTEN signaling axis].

The present study aimed to investigate the effect and mechanism of Bupleuri Radix-Paeoniae Radix Alba medicated plasma on HepG2 hepatoma cells by regulating the microRNA-1297(miR-1297)/phosphatase and tensin homologue deleted on chromosome 10(PTEN) signaling axis. Real-time quantitative PCR(RT-qPCR) was carried out to determine the mRNA levels of miR-1297 and PTEN in different hepatoma cell lines. The dual luciferase reporter assay was employed to verify the targeted interaction between miR-1297 and PTEN. The cell counting kit-8(CCK-8) was used to detect cell proliferation, and the optimal concentration and intervention time of the medicated plasma were determined. The cell invasion and migration were examined by Transwell assay and wound healing assay. Cell cycle distribution was detected by PI staining, and the apoptosis of cells was detected by Annexin V-FITC/PI double staining. The mRNA levels of miR-1297, PTEN, protein kinase B(Akt), and phosphatidylinositol 3-kinase(PI3K) were determined by RT-qPCR. Western blot was employed to determine the protein levels of PTEN, Akt, p-Akt, caspase-3, caspase-9, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). The results showed that HepG2 cells were the best cell line for subsequent experiments. The dual luciferase reporter assay confirmed that miR-1297 could bind to the 3'-untranslated region(3'UTR) in the mRNA of PTEN. The medicated plasma inhibited the proliferation of HepG2 cells, and the optimal intervention concentration and time were 20% and 72 h. Compared with the blank plasma, the Bupleuri Radix-Paeoniae Radix Alba medicated plasma, miR-1297 inhibitor, miR-1297 inhibitor + medicated plasma all inhibited the proliferation, invasion, and migration of HepG2 cells, increased the proportion of cells in the G_0/G_1 phase, decreased the proportion of cells in the S phase, and increased the apoptosis rate. The medicated plasma down-regulated the mRNA levels of miR-1297, PI3K, and Akt and up-regulated the mRNA level of PTEN. In addition, it up-regulated the protein levels of PTEN, Bax, caspase-3, and caspsae-9 and down-regulated the protein levels of p-Akt, p-PI3K, and Bcl-2. In conclusion, Bupleuri Radix-Paeoniae Radix Alba medicated plasma can inhibit the expression of miR-1297 in HepG2 hepatoma cells, promote the expression of PTEN, and negatively regulate PI3K/Akt signaling pathway, thereby inhibiting the proliferation and inducing the apoptosis of HepG2 cells.

[Mechanism of Kuntai Capsules in treatment of polycystic ovary syndrome rat models based on AGE-RAGE signal pathway].

This study aims to investigate the impact of Kuntai Capsules(KTC) on polycystic ovarian syndrome(PCOS) rat models and explore the underlying mechanism. Fifty female SD rats were randomly divided into five groups(10 rats in each group), including control group, model group, low-, medium-, and high-dose KTC group. Except for the control group, the other groups were injected with dehydroepiandrosterone(DHEA) combined with a high-fat diet(HFD) to induce the PCOS rat model for 28 days. 0.315, 0.63, and 1.26 g·kg~(-1)·d~(-1) KTC was dissolved in the same amount of normal saline and given to low-, medium-, and high-dose KTC groups by gavage. Both control group and model group were given the same amount of normal saline for 15 days. After administration, fasting blood glucose(FBG) was measured by a glucose meter. Fasting insulin(FINS), luteinizing hormone(LH), testosterone(T), and follicle-stimulating hormone(FSH) were detected by enzyme-linked immunosorbent assay(ELISA), and LH/FSH ratio and insulin resistance index(HOMA-IR) were calculated. The pathological morphology of ovarian tissue was observed by hematoxylin-eosin(HE) staining. The expression levels of collagen α type Ⅲ 1 chain(COL3A1), apoptotic factors Bax, and Bcl-2 were detected using Western blot and immunofluorescence. The mRNA expressions of COL3A1, Bax, and Bcl-2 in ovarian tissue were performed by real-time PCR(RT-PCR). The results show that compared with the control group, the body weight, serum levels of FBG, FINS, LH, T, LH/FSH, and HOMA-IR are higher in model group(P<0.05 or P<0.01), and the level of FSH is lower(P<0.05). In model group, a large number of white blood cells are found in the vaginal exfoliated cells, mainly in the interictal phase. There are more cystic prominences on the surface of the ovary. The thickness of the granular cell layer is reduced, and oocytes are absent. COL3A1 and Bax protein expression levels are increased(P<0.01), while Bcl-2 protein expression levels are decreased(P<0.05) in the ovarian tissue COL3A1 and Bax mRNA expression levels are increased in ovarian tissue(P<0.05). Compared with the model group, the body weight, FBG, FINS, LH, T, LH/FSH, and HOMA-IR in low-, medium-, and high-dose KTC groups are decreased(P<0.05 or P<0.01), while the levels of FSH in medium-, and high-dose KTC groups are increased(P<0.05 or P<0.01). Low-, medium-, and high-dose KTC groups gradually show a stable interictal phase. The surface of the ovary is smooth. Oocytes and mature follicles can be seen in ovarian tissue, and the thickness of the granular cell layer is increased. The expression level of COL3A1 protein decreases in low-and medium-dose KTC groups(P<0.05 or P<0.01), and that of Bax protein decreases in low-dose KTC group(P<0.05 or P<0.01), and the expression level of Bcl-2 protein increases in low-dose KTC group(P<0.01). The expression levels of COL3A1 and Bax mRNA decreased in the low-dose KTC group(P<0.05), while the expression levels of Bcl-2 mRNA increased(P<0.05). In summary, KTC can inhibit ovarian granulosa cell apoptosis and reduce follicular atresia by regulating the AGE-RAGE signaling pathway. It can promote insulin secretion, reduce blood sugar and body weight, restore serum hormone levels, improve symptoms of PCOS, alleviate morphological damage of the ovary, and restore ovarian function, which is of great value in the treatment of PCOS.

[Tanshinone â ¡_A inhibits ferroptosis via Nrf2 signaling pathway to protect liver in rats of non-alcoholic fatty liver disease].

This study investigated the protective effect of tanshinone Ⅱ_A(TSⅡ_A) on the liver in the rat model of non-alcoholic fatty liver disease(NAFLD) and the mechanism of TSⅡ_A in regulating ferroptosis via the nuclear factor E2-related factor 2(Nrf2) signaling pathway. The rat model of NAFLD was established with a high-fat diet for 12 weeks. The successfully modeled rats were assigned into model group, low-and high-dose TSⅡ_A groups, and inhibitor group, and normal control group was set. Enzyme-linked immunosorbent assay was employed to determine the content of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum of rats in each group. A biochemical analyzer was used to measure the content of aspartate aminotransferase(AST), alaninl aminotransferase(ALT), total cholesterol(TC), and triglycerides(TG). Hematoxylin-eosin(HE) staining was used to detect pathological damage in liver tissue. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling(TUNEL) was employed to examine the apoptosis of the liver tissue. Oil red O staining, MitoSOX staining, and Prussian blue staining were conducted to reveal lipid deposition, the content of reactive oxygen species(ROS), and iron deposition in liver tissue. Western blot was employed to determine the expression of Nrf2, heme oxygenase-1(HO-1), glutathione peroxidase 4(GPX4), ferroptosis suppressor protein 1(FSP1), B cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) in the liver tissue. The result showed that TSⅡ_A significantly reduced the content of MDA, AST, ALT, TC, and TG in the serum, increased the activity of SOD, decreased the apoptosis rate, lipid deposition, ROS, and iron deposition in the liver tissue, up-regulated the expression of Nrf2, HO-1, FSP1, GPX, and Bcl-2, and inhibited the expression of Bax in the liver tissue of NAFLD rats. However, ML385 partially reversed the protective effect of TSⅡ_A on the liver tissue. In conclusion, TSⅡ_A could inhibit ferroptosis in the hepatocytes and decrease the ROS and lipid accumulation in the liver tissue of NAFLD rats by activating the Nrf2 signaling pathway.

Cold atmospheric plasma attenuates skin cancer via ROS induced apoptosis.

BACKGROUND: Cold atmospheric plasma (CAP) has been widely used in biomedical research, especially in vitro cancer therapy. Cutaneous squamous cell carcinoma (CSCC) is a malignant tumor originating from epidermal keratinocytes. However, the mechanism of CAP therapy on CSCC remains unclear. METHODS AND RESULTS: The animal models of CSCC induced by 7,12-dimethylbenz(a) anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) were constructed. For the CAP treatment group, after each TPA application, CAP was administered for 3 min twice weekly after drying. HE staining were used to detect the pathological status of tumor tissue in each group. The levels of PCNA, Bcl-2, Bax, MMP2 and MMP9 were evaluated by western blot and qPCR. TUNEL staining were used to detect apoptosis in tumor tissues. In vivo, serum samples were used for ELISA of total ROS. MTT assay was used to detect the viability of A431 cells. Western blot and qPCR were used to detect the levels of PCNA, Bcl-2, Bax, MMP2 and MMP9 in A431 cells. A431 cell proliferation was examined by colony formation assay. The proportions of apoptosis of A431 cells were detected by flow cytometry. Transwell assessed the ability of A431 cells migration and proliferation. We found that CAP could induce skin cancer cells apoptosis and inhibit the progress of skin cancer. Through experiments in vitro, reactive oxygen species (ROS) generated by N-acetylcysteine (NAC) and CAP inhibited the proliferation and migration of A431 skin cancer cells while promoting apoptosis. CONCLUSIONS: These evidences suggest the protective effect of CAP in CSCC, and CAP has the potential clinical application of CSCC.