Individual cytotoxicity of three major type A trichothecene, T-2, HT-2, and diacetoxyscirpenol in human Jurkat T cells.

Mycotoxins are toxic, fungal secondary metabolites that contaminate agricultural commodities, food, and feed. Among them, T-2, HT-2, and diacetoxyscirpenol (DAS; the major type A trichothecene) are primarily produced from Fusarium species. These mycotoxins exert numerous toxicological effects in animals and humans, such as dermatotoxicity, haematotoxicity, hepatotoxicity, nephrotoxicity, neurotoxicity, and immunotoxicity. In the present study, human Jurkat T cells were used as a model to investigate apoptotic cell death induced by T-2, HT-2, and DAS. The results showed that T-2, HT-2, and DAS decreased cell viability and increased production of Reactive Oxygen Species in a time- and dose-dependency. Based on their IC50 values, they could be ranked in decreasing order of cytotoxicity as T-2 > HT-2 > DAS. All tested mycotoxins caused DNA fragmentation, up-regulated cytochrome C, caspase 3, and caspase 9 mRNA levels, and down-regulated the relative expression of Bcl-2 and caspase 8. The effects of these trichothecenes on apoptosis were determined based on flow cytometry. At the IC50 concentrations, the percentages of apoptotic cells were significantly higher than for the controls. Taken together, these data suggested that T-2, HT-2, and DAS could induce apoptosis through the mitochondrial apoptotic pathway.

Resveratrol promotes the differentiation of human umbilical cord mesenchymal stem cells into esophageal fibroblasts via AKT signaling pathway.

Objectives: Resveratrol has been implicated in the differentiation and development of human umbilical cord mesenchymal stem cells. The differentiation of into esophageal fibroblasts is a promising strategy for esophageal tissue engineering. However, the pharmacological effect and underlying mechanism of resveratrol on human umbilical cord mesenchymal stem cells differentiation are unknown. Here, we investigated the effects and mechanism of resveratrol on the differentiation of human umbilical cord mesenchymal stem cells. Methods: Using a transwell-membrane coculture system to culture human umbilical cord mesenchymal stem cells and esophageal fibroblasts, we examined how resveratrol act on the differentiation of human umbilical cord mesenchymal stem cells. Immunocytochemistry, Sirius red staining, quantitative real-time PCR, and Western blotting were performed to examine collagen synthesis and possible signaling pathways in human umbilical cord mesenchymal stem cells. Results: We found that resveratrol promoted collagen synthesis and AKT phosphorylation. However, co-treatment of cells with resveratrol and the PI3K inhibitor LY294002 inhibited collagen synthesis and AKT phosphorylation. We demonstrated that resveratrol down-regulated the expression of IL-6, TGF-ß, caspase-9, and Bax by activating the AKT pathway in human umbilical cord mesenchymal stem cell. Furthermore, resveratrol inhibited phosphorylated NF-ĸB in human umbilical cord mesenchymal stem cells. Conclusion: Our data suggest that resveratrol promotes the differentiation of human umbilical cord mesenchymal stem cells into fibroblasts. The underlying mechanism is associated with the downregulation of IL-6 and TGF-ß via the AKT pathway and by inhibiting the NF-ĸB pathway. Resveratrol may be useful for esophageal tissue engineering.

In Vitro Inhibition of Colorectal Cancer Gene Targets by Withania somnifera L. Methanolic Extracts: A Focus on Specific Genome Regulation.

An approach that shows promise for quickening the evolution of innovative anticancer drugs is the assessment of natural biomass sources. Our study sought to assess the effect of W. somnifera L. (WS) methanolic root and stem extracts on the expression of five targeted genes (cyclooxygenase-2, caspase-9, 5-Lipoxygenase, B-cell lymphoma-extra-large, and B-cell lymphoma 2) in colon cancer cell lines (Caco-2 cell lines). Plant extracts were prepared for bioassay by dissolving them in dimethyl sulfoxide. Caco-2 cell lines were exposed to various concentrations of plant extracts, followed by RNA extraction for analysis. By explicitly relating phytoconstituents of WS to the dose-dependent overexpression of caspase-9 genes and the inhibition of cyclooxygenase-2, 5-Lipoxygenase, B-cell lymphoma-extra-large, and B-cell lymphoma 2 genes, our novel findings characterize WS as a promising natural inhibitor of colorectal cancer (CRC) growth. Nonetheless, we recommend additional in vitro research to verify the current findings. With significant clinical benefits hypothesized, we offer WS methanolic root and stem extracts as potential organic antagonists for colorectal carcinogenesis and suggest further in vivo and clinical investigations, following successful in vitro trials. We recommend more investigation into the specific phytoconstituents in WS that contribute to the regulatory mechanisms that inhibit the growth of colon cancer cells.

Investigation of apoptotic effects of Cucurbitacin D, I, and E mediated by Bax/Bcl-xL, caspase-3/9, and oxidative stress modulators in HepG2 cell line.

Cucurbitacins, natural compounds highly abundant in the Cucurbitaceae plant family, are characterized by their anticancer, anti-inflammatory, and hepatoprotective properties. These compounds have potential as therapeutic agents in the treatment of liver cancer. This study investigated the association of cucurbitacin D, I, and E (CuD, CuI, and CuE) with the caspase cascade, Bcl-2 family, and oxidative stress modulators in the HepG2 cell line. We evaluated the antiproliferative effects of CuD, CuI, and CuE using the MTT assay. We analyzed Annexin V/PI double staining, cell cycle, mitochondrial membrane potential, and wound healing assays at different doses of the three compounds. To examine the modulation of the caspase cascade, we determined the protein and gene expression levels of Bax, Bcl-xL, caspase-3, and caspase-9. We evaluated the total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase (SOD), glutathione (GSH), Total, and Native Thiol levels to measure cellular redox status. CuD, CuI, and CuE suppressed the proliferation of HepG2 cells in a dose-dependent manner. The cucurbitacins induced apoptosis by increasing caspase-3, caspase-9, and Bax activity, inhibiting Bcl-xL activation, causing loss of ΔΨm, and suppressing cell migration. Furthermore, cucurbitacins modulated oxidative stress by increasing TOS levels and decreasing SOD, GSH, TAS, and total and native Thiol levels. Our findings suggest that CuD, CuI, and CuE exert apoptotic effects on the hepatocellular carcinoma cell line by regulating Bax/Bcl-xL, caspase-3/9 signaling, and causing intracellular ROS increase in HepG2 cells.

The gut microbiota contributes to the infection of bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and causes significant economic losses. Gut microbiota is a large microbial community and has a variety of biological functions. However, whether there is a correlation between gut microbiota and BVDV infection and what kind of relation between them have not been reported. Here, we found that gut microbiota composition changed in normal mice after infecting with BVDV, but mainly the low abundance microbe was affected. Interestingly, BVDV infection significantly reduced the diversity of gut microbiota and changed its composition in gut microbiota-dysbiosis mice. Furthermore, compared with normal mice of BVDV infection, there were more viral loads in the duodenum, jejunum, spleen, and liver of the gut microbiota-dysbiosis mice. However, feces microbiota transplantation (FMT) reversed these effects. The data above indicated that the dysbiosis of gut microbiota was a key factor in the high infection rate of BVDV. It is found that the IFN-I signal was involved by investigating the underlying mechanisms. The inhibition of the proliferation and increase in the apoptosis of peripheral blood lymphocytes (PBL) were also observed. However, FMT treatment reversed these changes by regulating PI3K/Akt, ERK, and Caspase-9/Caspase-3 pathways. Furthermore, the involvement of butyrate in the pathogenesis of BVDV was also further confirmed. Our results showed for the first time that gut microbiota acts as a key endogenous defense mechanism against BVDV infection; moreover, targeting regulation of gut microbiota structure and abundance may serve as a new strategy to prevent and control the disease.IMPORTANCEWhether the high infection rate of BVDV is related to gut microbiota has not been reported. In addition, most studies on BVDV focus on in vitro experiments, which limits the study of its prevention and control strategy and its pathogenic mechanism. In this study, we successfully confirmed the causal relationship between gut microbiota and BVDV infection as well as the potential molecular mechanism based on a mouse model of BVDV infection and a mouse model of gut microbiota dysbiosis. Meanwhile, a mouse model which is more susceptible to BVDV provided in this study lays an important foundation for further research on prevention and control strategy of BVDV and its pathogenesis. In addition, the antiviral effect of butyrate, the metabolites of butyrate-producing bacteria, has been further revealed. Overall, our findings provide a promising prevention and control strategy to treat this infectious disease which is distributed worldwide.

Ruxolitinib induces apoptosis and pyroptosis of anaplastic thyroid cancer via the transcriptional inhibition of DRP1-mediated mitochondrial fission.

Anaplastic thyroid carcinoma (ATC) has a 100% disease-specific mortality rate. The JAK1/2-STAT3 pathway presents a promising target for treating hematologic and solid tumors. However, it is unknown whether the JAK1/2-STAT3 pathway is activated in ATC, and the anti-cancer effects and the mechanism of action of its inhibitor, ruxolitinib (Ruxo, a clinical JAK1/2 inhibitor), remain elusive. Our data indicated that the JAK1/2-STAT3 signaling pathway is significantly upregulated in ATC tumor tissues than in normal thyroid and papillary thyroid cancer tissues. Apoptosis and GSDME-pyroptosis were observed in ATC cells following the in vitro and in vivo administration of Ruxo. Mechanistically, Ruxo suppresses the phosphorylation of STAT3, resulting in the repression of DRP1 transactivation and causing mitochondrial fission deficiency. This deficiency is essential for activating caspase 9/3-dependent apoptosis and GSDME-mediated pyroptosis within ATC cells. In conclusion, our findings indicate DRP1 is directly regulated and transactivated by STAT3; this exhibits a novel and crucial aspect of JAK1/2-STAT3 on the regulation of mitochondrial dynamics. In ATC, the transcriptional inhibition of DRP1 by Ruxo hampered mitochondrial division and triggered apoptosis and GSDME-pyroptosis through caspase 9/3-dependent mechanisms. These results provide compelling evidence for the potential therapeutic effectiveness of Ruxo in treating ATC.

Effect of photodynamic therapy mediated by hematoporphyrin derivatives on small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells.

To investigate the effects of photodynamic therapy (PDT) mediated by hematoporphyrin derivatives (HPD) on the proliferation of small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells. H446 cells and BEAS-2B cells were cultured in vitro with different concentrations of HPD(0, 5, 10, 12, 15, 20 µg/mL) for 4 h, and then irradiated with 630 nm laser with different energy densities (0, 25, 50, 75, 100 mW/cm2). Cell viability of H446 cells and BEAS-2B cells were detected by CCK8 assay. The cell apoptosis was observed with Annexin V-FTTC/PI double staining and Hoechst 33258. The RT-PCR examination was applied to detect the transcriptional changes of the mRNA of Bax、Bcl-2, and Caspase-9. The results of CCK8 showed that when the HPD was 15 µg/mL and the laser power density reached 50 mW/cm2, the cell viability was significantly decreased compared with the black control group. Hoechst 33258 staining showed that with the increase of HPD concentration, the cell density was reduced, and apoptotic cells increased. Flow cytometry assay revealed that the apoptotic rates of the HPD-PDT group of H446 cells and BEAS-2B cells were significantly different from those of the blank control group. The RT-PCR examination showed that the expression levels of Bax and Caspase-9 mRNA in the HPD-PDT group were up-regulated, while the expression levels of Bcl-2 mRNA were down-regulated significantly. HPD-PDT can inhibit H446 cells and BEAS-2B cells growth. The mechanism may be related to up-regulating the expression levels of Bax and Caspase-9 mRNA and down-regulating the expression levels of Bcl-2 mRNA.

The role of DRP1 mediated mitophagy in HT22 cells apoptosis induced by silica nanoparticles.

Silica nanoparticles (SiNPs) are widely used in the biomedical field and can enter the central nervous system through the blood-brain barrier, causing damage to hippocampal neurons. However, the specific mechanism remains unclear. In this experiment, HT22 cells were selected as the experimental model in vitro, and the survival rate of cells under the action of SiNPs was detected by MTT method, reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and adenosine triphosphate (ATP) were tested by the kit, the ultrastructure of the cells was observed by transmission electron microscope, membrane potential (MMP), calcium ion (Ca2+) and apoptosis rate were measured by flow cytometry, and the expressions of mitochondrial functional protein, mitochondrial dynein, mitochondrial autophagy protein as well as apoptosis related protein were detected by Western blot. The results showed that cell survival rate, SOD, CAT, GSH-Px, ATP and MMP gradually decreased with the increase of SiNPs concentration, while intracellular ROS, Ca2+, LDH and apoptosis rate increased with the increase of SiNPs concentration. In total cellular proteins,the expressions of mitochondrial functional proteins VDAC and UCP2 gradually increased, the expression of mitochondrial dynamic related protein DRP1 increased while the expressions of OPA1 and Mfn2 decreased. The expressions of mitophagy related proteins PINK1, Parkin and LC3Ⅱ/LC3Ⅰ increased and P62 gradually decreased, as well as the expressions of apoptosis related proteins Apaf-1, Cleaved-Caspase-3, Caspase-3, Caspase-9, Bax and Cyt-C. In mitochondrial proteins, the expressions of mitochondrial dynamic related proteins DRP1 and p-DRP1 were increased, while the expressions of OPA1 and Mfn2 were decreased. Expressions of mitochondrial autophagy associated proteins PINK1, Parkin, LC3II/LC3I increased, P62 decreased gradually, as well as the expressions of apoptosis related proteins Cleaved-Caspase-3, Caspase-3, and Caspase-9 increased, and Cyt-C expressions decreased. To further demonstrate the role of ROS and DRP1 in HT22 cell apoptosis induced by SiNPs, we selected the ROS inhibitor N-Acetylcysteine (NAC) and Dynamin-related protein 1 (DRP1) inhibitor Mdivi-1. The experimental results indicated that the above effects were remarkably improved after the use of inhibitors, further confirming that SiNPs induce the production of ROS in cells, activate DRP1, cause excessive mitochondrial division, induce mitophagy, destroy mitochondrial function and eventually lead to apoptosis.

MicroRNA-129-1-3p protects chicken granulosa cells from cadmium-induced apoptosis by down-regulating the MCU-mediated Ca2+ signaling pathway.

Cadmium (Cd) is known as a female reproductive toxicant. Our previous study has shown that Cd can influence the proliferation and cell cycle of granulosa cells and induce apoptosis. MicroRNAs (miRNAs) play an important role in the regulation of Cd-induced granulosa cell damage in chickens. However, the mechanism remains unclear. In this study, we investigated the mechanisms by which microRNA-129-1-3p (miR-129-1-3p) regulates Cd-induced cytotoxicity in chicken granulosa cells. As anticipated, exposure to Cd resulted in the induction of oxidative stress in granulosa cells, accompanied by the downregulation of antioxidant molecules and/or enzymes of Nrf2, Mn-SOD, Cu-Zn SOD and CAT, and the upregulation of Keap1, GST, GSH-Px, GCLM, MDA, hydrogen peroxide and mitochondrial reactive oxygen species (mtROS). Further studies found that Cd exposure causes mitochondrial calcium ions (Ca2+) overload, provoking mitochondrial damage and apoptosis by upregulating IP3R, GRP75, VDAC1, MCU, CALM1, MFF, caspase 3, and caspase 9 gene and/or protein expressions and mitochondrial Ca2+ levels, while downregulating NCX1, NCLX and MFN2 gene and/or protein expressions and mitochondrial membrane potential (MMP). The Ca2+ chelator BAPTA-AM or the MCU inhibitor MCU-i4 significantly rescued Cd-induced mitochondrial dysfunction, thereby attenuating apoptosis. Additionally, a luciferase reported assay and western blot analysis confirmed that miR-129-1-3p directly target MCU. MiR-129-1-3p overexpression almost completely inhibited protein expression of MCU, increased the gene and protein expressions of NCLX and MFN2 downregulated by Cd, and attenuated mitochondrial Ca2+ overload, MMP depression and mitochondria damage induced by Cd. Moreover, the overexpression of miR-129-1-3p led to a reduction in mtROS and cell apoptosis levels, and a suppression of the gene and protein expressions of caspase 3 and caspase 9. As above, these results provided the evidence that IP3R-MCU signaling pathway activated by Cd plays a significant role in inducing mitochondrial Ca2+ overload, mitochondrial damage, and apoptosis. MiR-129-1-3p exerts a protective effect against Cd-induced granulosa cell apoptosis through the direct inhibition of MCU expression in the ovary of laying hens.

Protective effects of vitamin E against acrylamide-induced hepatotoxicity and nephrotoxicity from fetal development to adulthood: Insights into Akt/NF-κB and Bcl-xL/Bax signaling pathways.

Acrylamide (ACR), a toxin present in fried and baked carbohydrate-rich foods, is known to cause liver and kidney damage. This study aimed to investigate the mechanisms of oxidative stress, inflammation, and apoptosis that contribute to liver and kidney damage induced by chronic administration of ACR. Additionally, the effectiveness of vitamin E in mitigating these toxic effects was examined. The study initially involved dividing 40 pregnant rats into four groups. After lactation, the research continued with male offspring rats from each group. The offspring rats were divided into Control, Vitamin E, ACR, and ACR + Vitamin E groups. Following ACR administration, liver and kidney function tests were performed on serum samples. Biochemical analyses, evaluation of inflammation markers, histopathological examination, and assessment of protein levels of Akt/IκBα/NF-κB, Bax, Bcl-xL, and Caspase-9 were conducted on liver and kidney tissues. The analysis demonstrated that ACR adversely affected liver and kidney function, resulting in oxidative stress, increased inflammation, and elevated apoptotic markers. Conversely, administration of vitamin E positively impacted these parameters, restoring them to control levels. Based on the results, the mechanism of ACR's action on oxidative stress and inflammation-induced liver and kidney damage may be associated with the activation of apoptotic markers such as Bax and Caspase-9, as well as the Akt/IκBα/NF-κB signaling pathway. Consequently, the protective properties of vitamin E establish it as an essential vitamin for the prevention or mitigation of various ACR-induced damages.

Dexpanthenol exhibits antiapoptotic and anti-inflammatory effects against nicotine-induced liver damage by modulating Bax/Bcl-xL, Caspase-3/9, and Akt/NF-κB pathways.

Chronic tobacco use can lead to liver damage and inflammation due to the accumulation of various toxins in the body. This study aimed to investigate the correlation between the molecular mechanisms of nicotine-induced liver injury, the caspase cascade, and the Akt/NF-κB signaling pathway, as well as the protective effects of dexpanthenol (DEX). Male rats were subjected to intraperitoneal injections of nicotine at a concentration of 0.5 mg/kg/day and/or DEX at a concentration of 500 mg/kg/day for 8 weeks. After the treatment period, liver function tests were conducted on serum samples, and tissue samples were analyzed for protein levels of Akt, NF-κB, Bax, Bcl-xL, Caspase-3, and Caspase-9, along with histopathological changes. Additionally, assessments of oxidative stress markers and proinflammatory cytokines were carried out. Nicotine administration led to elevated levels of IL-6, IL-1ß, MDA, TOS, and oxidative stress index, accompanied by decreased TAS levels. Moreover, nicotine exposure reduced the p-Akt/Akt ratio, increased NF-κB, Bax, Caspase-3, and Caspase-9 protein levels, and decreased the antiapoptotic protein Bcl-xL levels. DEX treatment significantly mitigated these effects, restoring the parameters to levels comparable to those of the control group. Nicotine-induced liver injury resulted in oxidative stress, inflammation, and apoptosis, mediated by Bax/Bcl-xL, Caspase-3, Caspase-9, and Akt/NF-κB pathways. Conversely, DEX effectively attenuated nicotine-induced liver injury by modulating apoptosis through NF-κB, Caspase-3, Caspase-9, Bax inhibition, and Bcl-xL activation.

Lentivirus-based shRNA of Caspase-3 gene silencing inhibits chondrocyte apoptosis and delays the progression of surgically induced osteoarthritis.

Chondrocyte apoptosis is an important pathological feature of osteoarthritis (OA). Excessive apoptosis of chondrocytes disrupts the dynamic balance of cell proliferation and apoptosis, with a marked reduction in chondrocytes and cartilage matrix disintegration, which represents the main pathology of OA. Caspases, especially Caspase-3, play a central role in cell apoptosis. In this study, a lentiviral vector was used to transduce caspase-3 short hairpin RNA (shRNA) into rat chondrocytes (RCs), and the apoptotic and phenotypic genes of RCs were analyzed using real-time PCR and western blotting in vitro. In addition, in vivo intra-articular injection of Caspase-3 shRNA lentivirus was performed in a surgically induced OA rat model. Our results showed that Caspase-3 gene silencing could down-regulate the TNF-α-mediated inflammatory gene expression of TNFR1, FADD, and IL-1ß, apoptotic gene expression of APAF1, Caspase-3, and Caspase-9, thereby attenuating the apoptotic pathway in vitro. Caspase-3 gene silencing also attenuated TNF-α-mediated decreased gene expression of ACAN, Col1-a1, and Col2-a1. Furthermore, Caspase-3 gene silencing could effectively reduce the OARSI score, and gene expression of Caspase-3, Caspase-9, MMP13, and TNF-α in a surgically induced OA rat model. Caspase-3 gene silencing may serve as a novel therapeutic strategy for cartilage injury and OA.

Dexpanthenol protects against nicotine-induced kidney injury by reducing oxidative stress and apoptosis through activation of the AKT/Nrf2/HO-1 pathway.

Dexpanthenol (DEX), a subtype of vitamin B5, plays an important role in anabolic reactions, cellular energy and regeneration in the body. Nicotine has been shown to induce kidney damage through the mechanisms of oxidative stress and apoptosis. The purpose of this study was to investigate the potential protective effects of DEX against nicotine-induced kidney damage through modulation of the AKT/Nrf2/HO-1 signaling pathway. Male rats were intraperitoneally administered with 0.5 mg/kg/day nicotine and/or 500 mg/kg/day DEX for 8 weeks. Following administration, renal function tests were conducted on serum samples, and histopathological examinations and analysis of oxidative stress markers and antioxidant enzymes were performed on tissue samples. Protein levels of Akt, Nrf-2, HO-1, Bcl-xL, and Caspase-9 were also evaluated. Nicotine administration resulted in decreased protein levels of p-Akt, Nrf-2, HO-1, and Bcl-xL and increased Caspase-9 protein levels. In addition, nicotine administration caused an increase in MDA, TOS, and OSI levels and a decrease in GSH, GSH-Px, GST, CAT, SOD, and TAS levels. Additionally, BUN and Creatinine levels increased after nicotine administration. DEX administration positively regulated these parameters and brought them closer to control levels. Nicotine-induced kidney injury caused apoptosis and oxidative stress through Caspase-9 activation. DEX effectively prevented nicotine-induced kidney damage by increasing intracellular antioxidant levels and regulating apoptosis through Bcl-xL activation. These findings suggest that DEX has potential as a protective agent against nicotine-induced kidney damage.

A functional chicken-liver hydrolysate-based supplement ameliorates alcohol liver disease via regulation of antioxidation, anti-inflammation, and antiapoptosis.

Tons of broiler livers are produced yearly in Taiwan but always considered waste. Our team has successfully patented and characterized a chicken-liver hydrolysate (CLH) with several biofunctions. Chronic alcohol consumption causes hepatosteatosis or even hepatitis, cirrhosis, and cancers. This study was to investigate the hepatoprotection of CLH-based supplement (GBHP01™) against chronic alcohol consumption. Results showed that GBHP01™ could reduce (p < .05) enlarged liver size, lipid accumulation/steatosis scores, and higher serum AST, ALT, γ-GT, triglyceride, and cholesterol levels induced by an alcoholic liquid diet. GBHP01™ reduced liver inflammation and apoptosis in alcoholic liquid-diet-fed mice via decreasing TBARS, interleukin-6, interleukin-1ß, and tumor necrosis factor-α levels, increasing reduced GSH/TEAC levels and activities of SOD, CAT and GPx, as well as downregulating CYP2E1, BAX/BCL2, Cleaved CASPASE-9/Total CASPASE-9 and Active CASPASE-3/Pro-CASPASE-3 (p < .05). Furthermore, GBHP01™ elevated hepatic alcohol metabolism (ADH and ALDH activities) (p < .05). In conclusion, this study prove the hepatoprotection of GBHP01™ against alcohol consumption.

Exploring the induction of endometrial epithelial cell apoptosis in clinical-type endometritis in yaks through the cyt-c/caspase-3 signaling axis.

Endometritis is a significant contributor to reduced productivity in yaks in Tibet, China. The Cyt-c/Caspase-3 signaling axis plays a crucial role in the mitochondrial pathway that triggers cell apoptosis due to endogenous factors. In this study, we examined the endometrial epithelial tissue of yaks with endometritis using pathological examination, immunohistochemical analysis, TUNEL staining, qRT-PCR, and Western blot. The results indicated significant changes in the apoptotic factors of the Cyt-c/Caspase-3 signaling axis. The expression levels of Bak1, Bax, Cyt-c, Apaf-1, Caspase-9, and Caspase-3 were significantly increased (P < 0.05), while the expression level of Bcl-2 was significantly decreased. Immunohistochemistry results revealed significant increase in Bak1, Bax, Cyt-c, Apaf-1, Caspase-9, and Caspase-3 expression in the cytoplasm compared to the healthy group, except for Bcl-2, which showed a significant decrease. Pathological section analysis demonstrated that clinical endometritis in yaks led to structural damage, bleeding, congestion, and inflammatory cell infiltration in the endometrial epithelium. Our study findings indicated that clinical endometritis in yaks can modulate apoptosis of endometrial epithelial cells via the Cyt-c/Caspase-3 signaling pathway, resulting in different levels of damage. This research is pioneering in exploring cell apoptosis induced by clinical endometritis in yaks, offering novel insights and potential strategies for the future prevention and treatment of endometritis in yaks.

Acetate attenuates cyclophosphamide-induced cardiac injury via inhibition of NF-kB signaling and suppression of caspase 3-dependent apoptosis in Wistar rats.

AIM: The goal of the current study was to examine the potential therapeutic effects of sodium acetate on cardiac toxicities caused by cyclophosphamide in Wistar rats. The possible involvement of NF-kB/caspase 3 signaling was also explored. MAIN METHODS: Thirty-two male Wistar rats were divided into four groups at random. (n = 8). The control animals received 0.5 mL of distilled water orally for 14 days, the acetate-treated group received 200 mg/kg/day of sodium acetate orally for 14 consecutive days, and cyclophosphamide-treated rats received 150 mg/kg /day of cyclophosphamide i.p. on day 8, while cyclophosphamide + acetate group received sodium acetate and cyclophosphamide as earlier stated. KEY FINDINGS: Results showed that cyclophosphamide-induced cardiotoxicity, which manifested as a marked drop in body and cardiac weights as well as cardiac weight/tibial length, increased levels of troponin, C-reactive protein, lactate, and creatinine kinase, and lactate dehydrogenase activities in the plasma and cardiac tissue. Histopathological examination also revealed toxic cardiac histopathological changes. These alterations were associated with a significant increase in xanthine oxidase and myeloperoxidase activities, uric acid, malondialdehyde, TNF-α, IL-1ß, NFkB, DNA fragmentation, and caspase 3 and caspase 9 activities in addition to a marked decline in Nrf2 and GSH levels, and SOD and catalase activities in the cardiac tissue. Acetate co-administration significantly attenuated cyclophosphamide cardiotoxicity by its antioxidant effect, preventing NFkB activation and caspase 9/caspase 3 signalings. SIGNIFICANCE: This study shows that acetate co-administration may have cardio-protective effects against cyclophosphamide-induced cardiotoxicity by inhibiting NF-kB signaling and suppressing caspase-3-dependent apoptosis.

Periploca forrestii Schltr. ameliorate liver injury caused by fluorosis in rat.

To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.

NK-derived exosome miR-1249-3p inhibits Mycobacterium tuberculosis survival in macrophages by targeting SKOR1.

Murine Natural Killer cells were cultivated in vitro to isolate NK-derived exosomes. Subsequent quantification via qPCR confirmed enrichment of miR-1249-3p. Ana-1 murine macrophages were cultured in vitro and subsequently inoculated with Mycobacterium tuberculosis (MTB) strain H37Rv. NK-exo and NK-exo miR-1249-3p were separately applied to the infection model, followed by immunological assays conducted post-48-hour co-culture. Western blot analyses corroborated that NK-exo exhibited exosomal marker proteins Granzyme A (GzmA), Granzyme B (GzmB), and Perforin (PFN), alongside a notable enrichment of miR-1249-3p. Functionally, NK-exo augmented the expression levels of Caspase-9,-8, and -3, as well as PARP, while attenuating the expression of NLRP3, ASC, and Cleaved-Caspase-1. Furthermore, qPCR demonstrated an up-regulation of Caspase-9, -8, and -3, along with pro-apoptotic factors Bax and Bid, and a concomitant down-regulation of the anti-apoptotic factor Bcl-2. The expression levels of inflammatory markers ASC, NLRP3, Cleaved-Caspase-1, and IL-1ß were concomitantly decreased. ELISA findings indicated diminished levels of TNF-α and ROS secretion. NK-exo miR-1249-3p specifically targeted and attenuated the expression of SKOR-1, engendering up-regulation of apoptosis-associated proteins and down-regulation of inflammation-related proteins, consequently affecting cellular fate.Our empirical evidence substantiates that NK-exo induces macrophage apoptosis, thereby mitigating MTB survival. Furthermore, NK-exo miR-1249-3p directly targets and inhibits SKOR-1 expression, leading to macrophage apoptosis and consequently hampering the proliferation of MTB. The data implicate the potential therapeutic relevance of NK-exo and miR-1249-3p in managing drug-resistant tuberculosis.

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

Luteolin Pretreatment Ameliorates Myocardial Ischemia/Reperfusion Injury by lncRNA-JPX/miR-146b Axis.

Background: In the present study, we aimed to find out whether luteolin (Lut) pretreatment could ameliorate myocardial ischemia/reperfusion (I/R) injury by regulating the lncRNA just proximal to XIST (JPX)/microRNA-146b (miR-146b) axis. Methods: We established the models in vitro (HL-1 cells) and in vivo (C57BL/6J mice) to certify the protection mechanism of Lut pretreatment on myocardial I/R injury. Dual luciferase reporter gene assay was utilized for validating that JPX could bind to miR-146b. JPX and miR-146b expression levels were determined by RT-qPCR. Western blot was utilized to examine apoptosis-related protein expression levels, including cleaved caspase-9, caspase-9, cleaved caspase-3, caspase-3, Bcl-2, Bax, and BAG-1. Apoptosis was analyzed by Annexin V-APC/7-AAD dualstaining, Hoechst 33342 staining, as well as flow cytometry. Animal echocardiography was used to measure cardiac function (ejection fraction (EF) and fractional shortening (FS) indicators). Results: miR-146b was demonstrated to bind and recognize the JPX sequence site by dual luciferase reporter gene assay. The expression level of miR-146b was corroborated to be enhanced by H/R using RT-qPCR (P < 0.001 vs. Con). Moreover, JPX could reduce the expression of miR-146b, whereas inhibiting JPX could reverse the alteration (P < 0.001 vs. H/R, respectively). Western blot analysis demonstrated that Lut pretreatment increased BAG-1 expression level and Bcl-2/Bax ratio, but diminished the ratio of cleaved caspase 9/caspase 9 and cleaved caspase 3/caspase 3 (P < 0.001 vs. H/R, respectively). Moreover, the cell apoptosis change trend, measured by Annexin V-APC/7-AAD dualstaining, Hoechst 33342 staining, along with flow cytometry, was consistent with that of apoptosis-related proteins. Furthermore, pretreatment with Lut improved cardiac function (EF and FS) (P < 0.001 vs. I/R, respectively), as indicated in animal echocardiography. Conclusion: Our results demonstrated that in vitro and in vivo, Lut pretreatment inhibited apoptosis via the JPX/miR-146b axis, ultimately improving myocardial I/R injury.