Caspase-3 Protein Expression Research Articles

Mechanism of intestinal injury induced by acute diquat poisoning in rats

To investigate the effects of diquat (DQ) on the expression of intestinal pyroptosis-related proteins and tight junction proteins in rats,and to analyze the role of pyroptosis in the intestinal injury of rats with acute DQ poisoning. A total of 36 Wistar male rats were randomly divided into control group, and 3 hours, 12 hours, 36 hours and 3 days exposure groups, with 6 rats in each group. Each exposure group was given 1/2 median lethal dose (LD50) of 115.5 mg/kg DQ by one-time gavage. The control group was given the same amount of normal saline by gavage. The control group was anesthetized at 3 hours after DQ gavage to take jejunal tissues; each exposure group was anesthetized at 3 hours, 12 hours, 36 hours, and 3 days after DQ gavage to take jejunal tissues, respectively. The general conditions of the rats were recorded. The pathological changes of jejunum tissue were observed by hematoxylin-eosin (HE) staining. The expression of intestinal pyroptosis-related proteins [NOD-like receptor protein 3 (NLRP3), cysteine aspartate-specific protease 1 (caspase-1), Gasdemin D (GSDMD)] in the intestinal tissues was observed by immunohistochemical staining. Western blotting was used to detect the expression of intestinal pyroptosis-related proteins and intestinal tight junction proteins (Occludin and Claudin-1). Light microscopy showed that pathological changes occurred in jejunum tissue at the early stage of exposure (3 hours), and the injury was the most serious in the 12 hours exposure group, with a large number of inflammatory cells infiltrating in the tissue, and the damage was significantly reduced after 3 days exposure. Immunohistochemical results showed that NLRP3, caspase-1 and GSDMD were expressed in the jejunal mucosa of the control group and the exposure groups, and the positive cells in the control group were less expressed with light staining. The expression of the above proteins in the exposed group was increased significantly and the staining was deep. Western blotting results showed that compared with the control group, the expression of NLRP3 protein in jejunum tissues of all groups was increased, with the most significant increase in the 36 hours group (NLRP3/β-actin: 1.47±0.06 vs. 0.43±0.14, P < 0.01). Compared with the control group, the expression of GSDMD protein in the 3 hours, 12 hours and 36 hours exposure groups increased, and the expression of GSDMD protein in the 3 hours and 12 hours exposure groups increased significantly (GSDMD/β-actin: 1.04±0.40, 1.25±0.15 vs. 0.65±0.25, both P < 0.05). The expression of caspase-1 protein was increased in 36 hours exposure group compared with the control group (caspase-1/β-actin: 1.44±0.34 vs. 0.98±0.19, P > 0.05). Compared with the control group, the expression of Occludin and Claudin-1 proteins in each exposure group decreased, and the expression of Occludin proteins was significantly decreased in the 3 hours, 12 hours, and 36 hours exposure groups decreased significantly (Occludin/β-actin: 0.74±0.17, 0.91±0.20, 0.79±0.23 vs. 1.41±0.08, all P < 0.05). Although the protein expression of Claudin-1 decreased in each exposure group, the difference was not statistically significant. The intestinal injury caused by acute DQ poisoning may be related to the activation of pyroptosis pathway of small intestinal cells and the reduction of the density of intercellular junctions.

Effect of Pistacia Terebinthus (terebinth) resin extract on aggressive breast cancer cells.

Breast cancer is one of the most common types of cancer causing high mortality rates among women worldwide. This study was aimed to evaluate the effect of Pistacia terebinthus (terebinth) resin extract (TRE) on the MDA-MB-231 breast cancer cell line. In the study, the cytotoxic dose of the resin extract in MDA-MB-231 cells was evaluated by MTS analysis. The effect of TRE on apoptosis was examined by Hoechst staining. Caspase-3 and cleaved caspase-3 protein expressions were determined by western blot analysis. Based on the outcomes of our MTS analysis, the IC50 dose of TRE was calculated at 56.54μg/mL during a 24-h application period. With Hoechst staining analysis, an increase was observed in cells that underwent apoptotic change at 10 and 100μg/ml TRE concentrations compared to the control. At 25 and 50μg/mL TRE concentrations, no apoptotic change was found in comparison to the control; however, a significant drop in the number of viable cells was observed because 200, 300, and 500μg/mL TRE concentrations were above the toxic dose. The caspase-3 protein expression level was significantly higher in cells treated with 100μg/ml TRE compared to the control group, while there was no significant change in cleaved caspase-3 protein expression. It was thought that P. terebinthus resin might cause cell death in MDA-MB-231 cells via caspase-independent apoptosis pathway or other cell death pathways, and it was concluded that it could be a supportive treatment for breast cancer treatment.

The Effect of Chrysin on the Distribution of Extracellular Matrix Proteins around Leydig Cells under Heat Stress Injury

Objective. In the testicular tissue stroma, matrix proteins such as laminin and fibronectin play a critical role in transmitting signals under normal and stressful conditions to Leydig cells. Heat stress is considered one of the stressful conditions in the testicular tissue. This study has investigated the protective effects of chrysin on the distribution of laminin and fibronectin around Leydig cells under heat stress. Methods. Rats were divided into four groups, including sham (A), heat stress (B), heat stress with chrysin treatment (50 mg/kg) (C), and heat stress with chrysin treatment (75 mg/kg) (D). At the end of the treatment period, on the 21st day, testosterone serum levels were evaluated, and the testes were also assessed to determine the changes in gene expression and distribution of laminin, fibronectin, and caspase-3 proteins. Results. Exposure to heat stress in Group B resulted in increased levels of caspase-3, decreased activity, height, and diameter of seminiferous tubules, and altered distribution of laminin and fibronectin proteins around the Leydig cells when compared to the sham group (p&lt;0.05). Treatment with chrysin 75 resulted in a significant decrease in caspase-3 levels, improved testosterone levels, and increased distribution of laminin and fibronectin around Leydig cells compared to group B (p&lt;0.05). In addition, the group treated with chrysin 75 showed improved seminiferous histological activity markers in comparison to group B (p&lt;0.05). Conclusion. Following heat stress, concurrent with disrupted steroidogenesis, an evident change happens in the distribution pattern of the extracellular matrix (ECM) proteins of the testis, which may indicate the transmission of stress signals from the ECM to the Leydig cells. Treatment of chrysin in 75 mg/kg dose can reduce the damage caused by heat stress on the ECM and Leydig cells.

MCC950 Attenuates Microglial NLRP3-Mediated Chronic Neuroinflammation and Memory Impairment in a Rat Model of Repeated Low-Level Blast Exposure.

Blast-induced traumatic brain injury is typically regarded as a signature medical concern for military personnel who are exposed to explosive devices in active combat zones. However, soldiers as well as law enforcement personnel may be repeatedly exposed to low-level blasts during training sessions with heavy weaponries as part of combat readiness. Service personnel who sustain neurotrauma from repeated low-level blast (rLLB) exposure do not display overt pathological symptoms immediately but rather develop mild symptoms including cognitive impairments, attention deficits, mood changes, irritability, and sleep disturbances over time. Recently, we developed a rat model of rLLB by applying controlled low-level blast pressures (≤ 70 kPa) repeated five times successively to mimic the pressures experienced by service members. Using this model, we assessed anxiety-like symptoms, motor coordination, and short-term memory as a function of time. We also investigated the role of the NLRP3 inflammasome, a complex involved in chronic microglial activation and pro-inflammatory cytokine interleukin (IL)-1β release, in rLLB-induced neuroinflammation. NLRP3 and caspase-1 protein expression, microglial activation, and IL-1β release were examined as factors likely contributing to these neurobehavioral changes. Animals exposed to rLLB displayed acute and chronic short-term memory impairments and chronic anxiety-like symptoms accompanied by increased microglial activation, NLRP3 expression, and IL-1β release. Treatment with MCC950, an NLRP3 inflammasome complex inhibitor, suppressed microglial activation, reduced NLRP3 expression and IL-1β release, and improved short-term memory deficits after rLLB exposure. Collectively, this study demonstrates that rLLB induces chronic neurobehavioral and neuropathological changes by increasing NLRP3 inflammasome protein expression followed by cytokine IL-1β release.

Potential Effects of Orally Ingesting Polyethylene Terephthalate Microplastics on the Mouse Heart.

Polyethylene terephthalate microplastics (PET MPs) are widespread in natural environment, and can enter organisms and accumulate in the body, but its toxicity has not been well studied. Therefore, in order to investigate the toxic effects of PET microplastics on mammals, this study investigated the toxic effects of PET MPs on ICR mice and H9C2 cells by different treatment groups. The results indicated the cardiac tissue of mice in the PET-H (50µg/mL) group showed significant capillary congestion, myocardial fiber breakage, and even significant fibrosis compared to the PET-C (control) group (P < 0.01). Results of the TUNEL assay demonstrated significant apoptosis in myocardial tissue in the PET-H and PET-M (5µg/mL) groups (P < 0.01). Meanwhile, Western blotting showed increased expression of the apoptosis-related protein Bax and decreased expression of PARP, caspase-3, and Bcl-2 proteins in both myocardial tissues and H9C2 cells. In addition, flow cytometry confirmed that PET MPs decreased the mitochondrial membrane potential and apoptosis in H9C2 cells; however, this trend was reversed by N-acetylcysteamine application. Moreover, PET MP treatment induced the accumulation of reactive oxygen species (ROS) in H9C2 cells, while the MDA level in the myocardial tissue was elevated, and the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were decreased (P < 0.01), indicating a change in the redox environment. In conclusion, PET MPs promoted cardiomyocyte apoptosis by inducing oxidative stress and activating mitochondria-mediated apoptotic processes, ultimately leading to myocardial fibrosis. This study provides ideas for the prevention of PET MP toxicity and promotes thinking about enhancing plastic pollution control.

Modulation of Apoptotic and Akt/PI3K/mTOR pathways to target Glioblastoma Cells using synthetic compound PGEA-AN.

To investigate the anticancer potential of a novel synthetic derivative of a naturally occurring diterpenoid against glioblastoma. The in vitro study was conducted at the Ojha Campus of Dow University of Health Sciences, Karachi, from February to December 2021, and comprised U87 cells. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the growth inhibitory effect of 16(R and S) - phenylamino-cleroda3, 13(14) Zdiene- 15, 16 olide and standard drug temozolomide against glioblastoma cells, and half-maximal inhibitory concentration was calculated. Microscopy and immunocytochemistry were used to investigate apoptotic morphology and active caspase-3 and B-cell lymphoma 2 (Bcl-2) expression. Quantitative real time polymerase chain reaction was used to investigate the expression of proliferation markers. Data was analysed using SPSS 21. Both the synthetic derivative and the standard drug significantly inhibited growth of U87 cells (p<0.001) with half-maximal inhibitory concentration of 19μM and 185μM, respectively. Apoptotic morphology and upregulation of active caspase-3 protein expression was observed in cells treated with half-maximal inhibitory concentration doses of both the synthetic derivative (p<0.05) and the standard drug (p<0.001), and Bcl-2 was downregulated in both the synthetic derivative (p<0.01) and the standard drug (p=0.05). However, no significant difference was observed in the expression of proliferation markers (p>0.05). The synthetic diterpene derivative PGEA-AN showed growth inhibitory actiity against glioblastoma.

Non-Contact Electric Field May Induced Higher CD4, CD8, Caspase-8, and Caspase-9 Protein Expression in Breast Tumor Tissue of Rats (Rattus norvegicus Berkenhout, 1769)

Non-contact electric field therapy has been studied as one of less invasive and safe alternative method in cancer therapy. Some studies reported that non-contact electric field therapy with low intensity (50 - 60 V/m) and medium frequency (150 kHz) could slower the rat’s breast tumor growth and did not cause significant damage on the rat's kidney and liver. However, further study needed to evaluate the potency of non-contact electric field to be developed into effective cancer therapy. Thus, we investigated the immune response of non-contact electric field therapy through the detection of CD4 and CD8 protein expression and the mechanism of apoptosis through the detection of caspase-8 and caspase-9 proteins on breast tumor tissue of rats. Twenty four rats were divided into 4 groups: 1. non-induction - non-therapy (NINT), 2. non-induction - therapy (NIT), 3. induction - non-therapy (INT), and 4. induction - therapy (IT). DMBA (7,12-dimethylbenz(α)anthracene) 20 mg/kg body weight was used to induced breast tumor formation. Rats with breast nodules that had reached 1 cm in size were exposed to an ECCT device with 150 kHz and 50 - 60 V/m electric fields. Further, the breast tissues were collected for routine histological and immunohistochemistry preparation for CD4, CD8, caspase 8, as well as caspase 9 detection. The data were statistically analyzed using Mann-Whitney U-Test method, with a significance level of p &lt; 0.05 using the SPSS 16.0 version. The scoring results were compared between the INT and IT groups. Our results showed that non-contact electric field therapy could suppress breast tumor growth and improved its histological structure. Interestingly, the breast tissue of IT group qualitatively had slightly more necrotic and apoptotic cells than that of INT group. Moreover, the IT group showed by higher CD4 and CD8 as well as higher caspase 8 and caspase 9 expression. Collecting all the data together, we concluded that non-contact electric field therapy potent to improve histological structure of breast tumor.

Urban Particulate Matter Triggers Meibomian Gland Dysfunction.

The meibomian gland (MG), as the largest modified sebaceous gland, is potentially damaged by urban particulate matter (UPM) based on epidemiological evidence, but the specific experimental mechanisms remain unknown. This study investigated the effects of UPM on MG dysfunction (MGD) in rodent models. Female C57BL/6J mice received eye drops containing UPM suspension or PBS for 14days. The proliferative capacity and progenitor of MG were evaluated by immunofluorescence. Cell apoptosis was confirmed by TUNEL assay, along with the analysis of caspase family expression. Lipid accumulation was visualized by Oil Red O staining and LipidTox staining. Ductal hyperkeratinization, neutrophil infiltration, and pyroptosis activation were detected through immunostaining. The relative gene expression and signaling pathway activation were determined by Western blot analysis. Administration of UPM caused MGD-like clinical signs, manifested as distinct corneal epithelial erosion, increased MG orifice occlusion, and glandular dropout. UPM exposure significantly induced progenitor loss, cellular apoptosis, and lipogenic disorder in MG, by reducing P63/Lrig1 expression and increasing cleaved caspase-8, -9, and -3 and meibum lipogenic protein (HMGCR/SREBP-1) expression. UPM-treated mice exhibited ductal hyperkeratinization and neutrophil recruitment. Simultaneously, pyroptosis was motivated, as indicated by the heightened expression of NLRP3 and the cleavage of caspase-1 and -4 and gasdermin D, as well as the increase in IL-1β and IL-18 downstream. The underlying pathological mechanisms of UPM involve the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB. These results provided direct evidence for the toxicity of UPM in MG. UPM-induced activation of pyroptosis and mitogen-activated protein kinase/nuclear factor-κB signaling pathway might account for the inflammatory MGD.

Effect of TRIM59 Expression Interference on Daunorubicin Chemosensitivity of Chronic Myeloid Leukemia K562 Cells and Its Mechanism

To investigate the effect of tripipartite motif 59 (TRIM59) expression interference on the chemosensitivity of daunorubicin (DNR) in chronic myeloid leukemia (CML) K562 cells and the related molecular mechanism. The expressions of TRIM59 mRNA in bone marrow tissues of patients with CML and K562 cells were detected by RT-qPCR. Liposome-based transfection technology was used to transfect TRIM59-specific siRNA (si-TRIM59) into K562 cells which then were treated with DNR. The proliferation and apoptosis of cells were detected by CCK-8 assay and flow cytometry, respectively, and the expressions of apoptosis-related protein and Wnt/β-catenin signaling pathway-related protein were detected by Western blot. Compared with the bone marrow tissue of CML patients at the time of initial treatment, the expression of TRIM59 mRNA in bone marrow tissue of CML patients at the time of chemotherapy resistance was significantly increased (P <0.05). Compared with control group, the cell proliferation inhibition rate and apoptosis rate in si-TRIM59 group and DNR group were significantly increased (P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased (P <0.05), while the expressions of Bcl-2, Wnt3α, GSK-3β protein and the ratio of p-β-catenin/β-catenin were significantly decreased (P <0.05). Compared with si-TRIM59 group and DNR group, the proliferation inhibition rate and apoptosis rate of si-TRIM59+DNR group were significantly increased (P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased, while the expression of Bcl-2, Wnt3α, GSK-3β protein and the ratio of p-β-catenin/β-catenin were significantly decreased (P <0.05). TRIM59 expression interference may enhance the chemosensitivity of K562 cells to DNR, and its mechanism may be related to the regulation of Wnt/β-catenin signaling pathway.

Protective effects of atractylenolide III on oxygen-glucose-deprivation/reperfusion-induced injury in HT22 cells.

Atractylenolide III (ATL III) is a natural bioactive compound, that possesses anti-inflammatory, antioxidant, and neuroprotective properties. However, whether ATL III can protect against neuronal injury induced by cerebral ischemia/reperfusion (I/R) have not yet been studied. This study aimed to investigate the protective effects of ATL III on neuronal injury using an oxygen-glucose deprivation/reperfusion (OGD/R) model in HT22 cells. Establishment of OGD/R model to induce HT22 cell injury in vitro. Cell viability, live-dead cell staining, oxidative stress levels, and pro-inflammatory cytokine levels were detected using kits. Cell apoptosis was observed by flow cytometry, and the expression of Bax, Bcl-2, and Caspase-3 proteins was detected by western blot. ATL III significantly alleviates OGD/R-induced cell injury, as evidenced by the increased cell viability and reduced apoptosis rate. ATL III increased the levels of superoxide dismutase (SOD) and glutathione (GSH), while reducing malondialdehyde (MDA), reactive oxygen species (ROS), and the levels of TNF-α, IL-1β, and IL-6. The protein expression of Bax and Caspase-3 was downregulated, while Bcl-2 expression was upregulated by ATL III. ATL III as a potential therapeutic agent for reducing neuronal injury by mitigating oxidative stress, apoptosis, and inflammation.

Lung-specific exosomes for doxorubicin delivery in lung adenocarcinoma therapy.

Doxorubicin (DOX) could be utilized to treat lung adenocarcinoma (LUAD), while dose-limiting cardiotoxicity limits its clinical utilization. MDA-MB-231 cell-derived exosomes show lung-specific organotropism features. In this study, we aimed to explore the potential of MDA-MB-231 cell-derived exosomes in DOX specific delivery to the lung. MDA-MB-231 cell-derived exosomes were coincubated with to construct for the doxorubicin delivery system (D-EXO). Exosomes labeled with fluorescein isothiocyanate were incubated with A549 cells or 293T cells, and the engulf and the mean intensity of the fluorescence were detected with immunofluorescence and flow cytometry assay. Cell viability was detected with cell counting kit-8 (CCK-8), and cell migration was determined by scratch test. The protein expression was detected by Western blot assay. A549 cell line-derived xenograft mouse model was constructed to examine the treatment effect of D-EXO. MDA-MB-231 cell-derived exosomes could be specially taken up by A549 cells with diminished cell viability but not engulfed by 293T cells. D-EXO inhibited A549 cell migration, and upregulated the protein expression of caspase 3 and cleaved caspase 3 expression, while did not show any inhibition on 293T cells. In vivo orthotopic xenotransplantation model indicated that D-EXO inhibited tumor growth characterized by diminished tumor weight and improved survival rate. No significant change in body weight was observed after the D-EXO treatment. In conclusion, D-EXO proposed in this study could be utilized to treat LUAD with lung-specific delivery effects to improve the survival rate.

Gaillardin inhibits autophagy and induces apoptosis in MCF-7 breast cancer cells by regulating JAK/STAT pathway.

Gaillardin is a potent anti-cancer sesquiterpene lactone found in Inula oculus-christi. The present study examined the effects of gaillardin on apoptosis and autophagy in the MCF-7 breast cancer cell line. The MTT assay was used to unravel the antiproliferative effects of gaillardin on MCF-7 cells. The expression of apoptosis-related genes including CASP3, BAX, BCL2, STAT3, and JAK2, and key markers of autophagy such as ATG1, ATG4, ATG5, ATG7, ATG12, BECN1, and MAP1LC3A were measured by real time-PCR method. The protein expression of Caspase 3, phosphorylated JAK2, phosphorylated STAT3, ATG1, ATG4, ATG5, ATG12, Beclin1, and LC-III was determined using western blotting. Gaillardin treatment significantly decreased the proliferation of MCF-7 cells with a parallel upregulation of the level of pro-apoptotic caspase-3 enzyme with no effect on Bax and Bcl2 expression. The levels of phosphorylated and active forms of JAK2 and STAT3 proteins were reduced following the treatment of MCF-7 cells with gaillardin. This sesquiterpene lactone com-pound considerably downregulated the levels of six autophagy markers, including ATG1, ATG4, ATG5, ATG12, Beclin1, and LC-III in MCF-7 cells. These data indicated the apoptosis-inducing activity of gaillardin in MCF-7 cells by a mechanism that inhibits the JAK/STAT signaling pathway. Further, autophagy inhibition was the other phenomenon caused by gaillardin in MCF-7 cells. These results can provide evidence to highlight the role of gaillardin as a novel therapeutic for the treatment of breast cancer.

Jianpi Antai formula prevents miscarriage by repressing M1 polarization of decidual macrophages through ubiquitination of NLRP3 mediated by MARCH7

Ethnopharmacological relevanceJianpi Antai Formula (JAF) is an ancient formula from He's gynecology, which has been used clinically for more than 30 years and has significant therapeutic effects on spontaneous abortion (SA). Both macrophage polarization and NLRP3 inflammasome correlate with the occurrence of SA in women with recurrent or threatened miscarriage. Whether JAF prevent SA via mediating activation of decidual macrophage (dMφ) and ubiquitination-associated degradation of NLRP3 remains uncertain. Aim of the studyThis study aimed to clarify the effects of JAF on pregnancy outcomes and dMφ polarization at the maternal-fetal interface in an SA mouse model, and use in vivo and invitro methods to explore whether JAF can inhibit M1 polarization of dMφ by up-regulating MARCH7-mediated NLRP3 ubiquitination, thereby preventing SA. Materials and methodsThe CBA/J × DBA/2 mating method was used to establish an SA model and the dMφs of SA mice were isolated and cultured. Th1-, Th2-, Th17- and Treg-related cytokine levels were evaluated using ELISA. qRT-PCR was used to detect the levels of M1/M2 macrophage-related cytokine mRNA in the decidua, and western blotting was used to detect the expression of NLRP3 inflammasome-related proteins in the decidua and placenta. The expression of M1/M2 markers of dMφ was detected using flow cytometry, ASC speck formation was observed using immunofluorescence, and the ubiquitination level of MARCH7-NLRP3 was detected using co-immunoprecipitation. ResultsJAF increased the survival rate of fetuses and the levels of estradiol and progesterone in SA model mice. It also reduced the serum Th1 and Th17-associated cytokine levels and decidual M1 macrophage-associated cytokine levels, while elevating the M2 macrophages in SA mice. NLRP3, caspase-1, ASC, and IL-1β protein expression in the decidua and placenta were also reduced. si-MARCH7 transfection reversed the effect of JAF on inhibiting the formation of the NLRP3 inflammasome and the activation of macrophages in dMφs of SA mice. ConclusionJAF could effectively prevent and treat SA by repressing M1 polarization of dMφs through NLRP3 ubiquitination and pyroptosis inhibition, which were mediated by MARCH7.

Antitumor Mechanism and Therapeutic Potential of Cordycepin Derivatives.

Cordycepin has good antitumor activity, but its clinical application is limited due to the easy deamination of N6 in structure. In this study, a large lipolysis group was introduced at the cordycepin N6 to improve the problem, cordycepin derivatives (3a-4c) were synthesized, and biological evaluation of compounds was studied. In this study, the vitro antitumor activity of the compounds against MCF7 cells, HepG2 cells and SGC-7901 cells was evaluated by MTT assay. In the results, compound 4a showed the most obvious inhibitory effect on MCF7 cells with an IC50 value of 27.57 ± 0.52 μM, which was much lower than cordycepin. Compound 4a showed high selectivity between MCF7 and normal MCF-10A cells. Further biological evaluation showed that compound 4a promoted apoptosis and blocked the cell cycle in the G0/G1 phase. Then, Western Blot was used to detect related apoptotic proteins. It was found that Compound 4a could down-regulate the expression of Bcl-2 protein and up-regulate the expression of p53, Bax, Caspase-3 and Caspase-9 proteins. The mitochondrial membrane potential decreased continuously and the positive expression rate decreased. It was speculated that compound 4a induced the apoptosis of MCF7 cells through the mitochondrial pathway.

Effect of the Novel Free Radical Scavenger 4′-Hydroxyl-2-Substituted Phenylnitronyl Nitroxide on Oxidative Stress, Mitochondrial Dysfunction and Apoptosis Induced by Cerebral Ischemia–Reperfusion in Rats

Mitochondrial dysfunction, which results in the overproduction of oxygen free radicals, is a crucial mechanism underlying cerebral ischemia–reperfusion injury. 4′-Hydroxyl-2-substituted phenylnitronyl nitroxide (HPN), which is an antioxidant and free radical scavenger, can effectively scavenge oxygen free radicals, suggesting its potential as a protective agent against cerebral ischemia–reperfusion injury. In this study, we investigated the effects of HPN on mitochondrial function and apoptosis following cerebral ischemia/reperfusion injury in rats. Healthy adult SD rats were chosen as the experimental subjects, and the rat ischemia/reperfusion injury model was generated using the modified Zea Longa method. The administration of HPN significantly enhanced the activity of endogenous antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT). Additionally, HPN effectively preserved the morphology and function of mitochondria, reduced the protein and gene expression of Caspase-3 and Bax, increased the protein and gene expression of Bcl-2, mitigated neuronal apoptosis, improved neurological deficits, and decreased the volume of cerebral infarction. Of interest, the protective effect on brain tissue was more evident with increasing doses of HPN. These findings indicate that HPN can serve as an effective protective agent against cerebral ischemia–reperfusion injury.

Th9 Cytokines Inhibit Proliferation, Promote Apoptosis, and Immune Escape in Thyroid Carcinoma Cells.

To investigate the regulatory effects of T helper 9 (Th9) cytokines on the proliferation, apoptosis and immune escape of thyroid cancer cells. The survival rate of human thyroid cancer cell line TPC-1 after treatment with 0, 1, 2.5, 5, 10, 20 ng/ml IL-9 (or IL-21) was determined by CCK-8 method and suitable concentrations of IL-9 and IL-21 were screened out. The TPC-1 cells cultured in vitro were randomly grouped into control group, IL-9 group, IL-21 group and IL-9+IL-21 group. After treatment with IL-9 and IL-21 factors, the proliferation and apoptosis of TPC-1 cells in each group were detected by CCK-8 method and flow cytometry, respectively. The flow cytometry was applied to detect the proportion of Th9 and activated CD8+ T cells in human peripheral blood lymphocytes co-cultured with TPC-1 in each group. The expression of TPC-1 and IL-9R and IL-21R protein in each group and human peripheral blood lymphocytes. Compared with the control group, the cell viability PCNA and Bcl-2 protein expression in TPC-1 cells were lower in the IL-9 group, IL-21 group and IL-9+IL-21 group (P<0.05). The apoptosis rate, proportions of Th9 and activated CD8+ T cells, killing rate of human peripheral blood lymphocytes, the expression of Bax and caspase-3 proteins in TPC-1 cells, the expression of TPC-1 and human peripheral blood lymphocytes IL-9R and IL-21R proteins were all higher (P<0.05) in IL-9+IL-21 group compared with the IL-9 group and the IL-21 group. The cell viability, PCNA and Bcl-2 protein expression in TPC-1 cells in the IL-9+IL-21 group were all lower (P<0.05). Th9 cytokines can promote the differentiation of Th9 cells and CD8+ T cells, enhance their lethality, reduce the immune escape of thyroid cancer cells, and then inhibit their proliferation and promote their apoptosis.

Naringin's Alleviation of the Inflammatory Response Caused by Actinobacillus pleuropneumoniae by Downregulating the NF-κB/NLRP3 Signalling Pathway.

Actinobacillus pleuropneumoniae (APP) is responsible for causing Porcine pleuropneumonia (PCP) in pigs. However, using vaccines and antibiotics to prevent and control this disease has become more difficult due to increased bacterial resistance and weak cross-immunity between different APP types. Naringin (NAR), a dihydroflavonoid found in citrus fruit peels, has been recognized as having significant therapeutic effects on inflammatory diseases of the respiratory system. In this study, we investigated the effects of NAR on the inflammatory response caused by APP through both in vivo and in vitro models. The results showed that NAR reduced the number of neutrophils (NEs) in the bronchoalveolar lavage fluid (BALF), and decreased lung injury and the expression of proteins related to the NLRP3 inflammasome after exposure to APP. In addition, NAR inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in porcine alveolar macrophage (PAMs), reduced protein expression of NLRP3 and Caspase-1, and reduced the secretion of pro-inflammatory cytokines induced by APP. Furthermore, NAR prevented the assembly of the NLRP3 inflammasome complex by reducing protein interaction between NLRP3, Caspase-1, and ASC. NAR also inhibited the potassium (K+) efflux induced by APP. Overall, these findings suggest that NAR can effectively reduce the lung inflammation caused by APP by inhibiting the over-activated NF-κB/NLRP3 signalling pathway, providing a basis for further exploration of NAR as a potential natural product for preventing and treating APP.

Phenolic-rich fraction of green tea attenuates histamine-mediated cardiopulmonary toxicity by inhibiting Cox-2/NF-κB signaling pathway and regulating oxidant/antioxidant balance

BackgroundHistamine (HIS) has a substantial impact on the development of numerous allergic disorders including asthma. Antihistamines mostly target histamine receptor-1 alone, so it is not entirely effective in the treatment of allergic diseases. In the current investigation, we examine the growing evidence for novel therapeutic strategies that aim to treat histamine-mediated cardiopulmonary toxicity with the phenolic-rich fraction of green tea (PRFGT).ResultsOur findings demonstrated that weekly ingestion of HIS to rats induced oxidant/antioxidant imbalance in both lung and heart homogenates. The histopathological examination demonstrated extensive interstitial pneumonia with progressive alveolar and bronchial damage in HIS receiving groups. Heart sections showed severe myocardial necrosis and hemorrhage. All lesions were confirmed by the immunohistochemical staining that demonstrated strong caspase-3, cyclooxygenase-2 (Cox-2), and tumor necrosis factor-α (TNF-α) protein expressions along with upregulation of the pulmonary m-RNA expression of TNF-α, nuclear factor kappa-B (NF-κB), and interleukin-1β (IL-1β) genes and cardiac levels of many apoptotic genes. Otherwise, the pretreatment of rats with PRFGT had the ability to alleviate all the aforementioned toxicological parameters and return the microscopic picture of both lung and heart sections to normal histology.ConclusionsWe concluded that PRFGT’s powerful antioxidant, anti-inflammatory, and anti-apoptotic properties can reduce cardiopulmonary toxicity caused by HIS. We recommended daily intake of green tea as a beverage or adding it to foods containing elevated levels of HIS to prevent its possible toxicity.

Modified Gexia-Zhuyu Tang inhibits gastric cancer progression by restoring gut microbiota and regulating pyroptosis

BackgroundGexia-Zhuyu Tang (GZT), a traditional Chinese medicine formula, is used to treat a variety of diseases. However, its roles in gastric cancer (GC) remain unclear.ObjectiveThe aim of this study was to explore the roles and underlying molecular mechanisms of modified GZT in GC.MethodsThe effects of modified GZT on GC were investigated by constructing mouse xenograft models with MFC cell line. The fecal samples from low-dose, high-dose, and without modified GZT treatment groups were collected for the 16S rRNA gene sequencing and fecal microbiota transplantation (FMT). Histopathological alterations of mice were evaluated using the hematoxylin–eosin (HE). Immunohistochemical (IHC) analysis with Ki67 and GSDMD was performed to measure tissue cell proliferation and pyroptosis, respectively. Proteins associated with pyroptosis, invasion, and metastasis were detected by Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to assess inflammation-related factors levels.ResultsModified GZT inhibited GC tumor growth and reduced metastasis and invasion-related proteins expression levels, including CD147, VEGF, and MMP-9. Furthermore, it notably promoted caspase-1-dependent pyroptosis, as evidenced by a dose-dependent increase in TNF-α, IL-1β, IL-18, and LDH levels, along with elevated protein expression of NLRP3, ASC, and caspase-1. Additionally, modified GZT increased species abundance and diversity of the intestinal flora. FMT assay identified that modified GZT inhibited GC tumor progression through regulation of intestinal flora.ConclusionsModified GZT treatment may promote pyroptosis by modulating gut microbiota in GC. This study identifies a new potential approach for the GC clinical treatment.

Baicalin Promotes Apoptosis of Human Medullary Breast Cancer via the ERK/p38 MAPK Pathway

Objectives The present study aims to examine the effects of baicalin on the human medullary breast carcinoma (MBC) cell line Bcap-37 and to determine whether baicalin regulates Bcap-37 cell apoptosis through the ERK/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Materials and Methods 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Transwell assay were utilized to measure the effects of baicalin on the proliferation and migration ability of Bcap-37 cells, respectively. Flow cytometer analysis was implied to detect the effects of baicalin on Bcap-37 cell apoptosis. Real-time quantitative polymerase chain reaction (RT-PCR) was conducted to observe the influence of baicalin on mRNA expressions of apoptosis-related genes. Western blot was executed to further explore the action of baicalin on apoptosis-related proteins. PD98059 (a specific inhibitor of ERK) and SB203580 (a specific inhibitor of p38 MAPK) were used to further clarify the intrinsic mechanism of baicalin regulating apoptosis in Bcap-37 cells. Results Compared with the control group, baicalin significantly inhibits the proliferation activity of Bcap-37 cells in a concentration- and time-dependent manner, with a p-value &lt; 0.05. The transwell assay indicated that the migration viability of cells decreased further, followed by the increased concentration of baicalin, and the p-value had a statistical difference. Besides, flow cytometry was conducted to assess the effects of baicalin on the early and late apoptosis rates of Bcap-37 cells, and results showed that baicalin highly promoted the apoptosis level both at the early and late stages with a statistical difference in a concentration-dependent manner ( p &lt; 0.05). Results of RT-PCR presented that, compared with the control group, baicalin significantly activated the mRNA expression of Bax, p38, and p-ERK1 and abolished the mRNA expression of Bcl-2 in every dosing group in a concentration-dependent way ( p &lt; 0.05). Western blot exhibited that, compared with the control group, baicalin promoted the protein expression of caspase-3, caspase-9, Bax, p38, P-ERK, and p53 while playing an opposite function to Bcl-2 in each dosing group ( p values have statistical differences). At last, PD98059 and SB203580 were applied to explore the potential mechanism of baicalin in apoptosis promotion. And results revealed that, compared with the group treated with baicalin alone, protein expression of Bax, p38, p-ERK, caspase-3, and caspase-9 was downregulated obviously in the group treated with both baicalin and PD98059 or SB203580 ( p &lt; 0.05). The p53 expression inhibition showed a difference only when compared to the group adding PD98059. Compared with the blank group, a statistical difference was only observed in Bcl-2 expression in the group treated with baicalin in combination with PD98059 or SB203580 ( p &lt; 0.05). Interestingly, a p53 expression difference was only displayed between the blank group and the group treated with baicalin and SB203580 ( p &lt; 0.05). Conclusion Our study observed that baicalin inhibits the proliferation and migration ability of Bcap-37 cells. In addition, baicalin induces cell apoptosis via the ERK/p38 MAPK signal pathway.