EXPLORING THE THERAPEUTIC POTENTIAL OF AM114: A BORONIC CHALCONE DERIVATIVE INDUCE APOPTOSIS AND SUPPRESS PROINFLAMMATORY CYTOKINES AND CHEMOKINES IN INTERLEUKIN-1? STIMULATED HUMAN THP-1 DERIVED MACROPHAGES

Objective: Chalcones and their derivatives display a wide range of pharmacological activities. This study examined the effects of AM114, a boronic-chalcone derivative, on human THP-1-derived macrophages with and without interleukin-1? (IL-1?) stimulation. Methods: AM114 and Aspirin-treated THP-1-derived macrophages underwent activation with or without interleukin-1?. The IC50 concentrations of AM114 and Aspirin were determined through an MTT test. Apoptosis was measured using various techniques, including staining with acridine orange/Ethidium bromide, Hoechst 33342, and rhodamine 123 assays. Caspase-3 activity was measured using the spectrofluorimetric technique, while DNA fragmentation was assessed via agarose gel electrophoresis. Pro-inflammatory cytokines such as interleukin-6 (IL-6) and chemokines like interleukin-8 (IL-8) were measured using enzyme-linked immunosorbent assays.Results: AM114 and Aspirin showed dose-dependent cytotoxic effects on THP-1 macrophages. Induction of apoptosis was detected in AM114-treated THP-1 macrophages activated with IL-1? compared to macrophages without IL-1?. The gradation of dye uptake, membrane blebbing, increased caspase-3 activity, and DNA fragmentation ensures the induction of apoptosis, which indicates the cell's morphological changes, biochemical processes, and mitochondrial activity. Treating AM114 in IL-1?-activated THP-1 macrophages significantly reduced pro-inflammatory cytokines (IL-6) and chemokines (IL-8), suggesting its anti-cytokine potential in inflammatory diseases.Conclusion: The study results emphasize that AM114 could act as an anti-inflammatory agent by triggering apoptosis and reducing the release of cytokines and chemokines in inflammatory conditions. As a result, it may be used as a therapeutic option for inflammatory diseases.

Benzydamine induces apoptosis in astrocytes in vitro / Benzadamina induz apoptose em astrócitos in vitro

Objective: the present work aims to evaluate the Benzydamine (BZD) effect on cell viability in astrocyte culture and investigated the death mechanism involved with its cytotoxic effect. Methods: in order to evaluate cell viability, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay was used, while flow cytometry was used to verify cell damage. The immunofluorescence assay was used to verify the expression of the marker's caspase-8, caspase9 and p65 subunit of Factor nuclear kappa B (NFκB). A statistical analysis for MTT assay and Flow Cytometry were made using ANOVA with Dunett's post-test; Student's t-test was made for the Immunofluorescence. Significance was set at p < 0.05. Results: the MTT reduction assay showed that BZD (3.1 to 100 µg/mL) caused a decrease in astrocytes viability. The flow cytometry showed that the cytotoxic effect of BZD was caused by the activation of the apoptotic death pathway, evidenced by the externalization of phosphatidylserine. The immunofluorescence revealed an increase in caspase-8 expression and no alteration in caspase-9 expression, demonstrating that there was an activation of the extrinsic pathway of apoptosis. The mean inhibitory concentration (IC50) of BZD (26.13 µg/mL) also caused an increase in NFκB p65 expression. Conclusion: taken together, the results of the present study suggest that BZD has a cytotoxic effect on astrocyte cells, and this effect comes from its ability to activate the extrinsic apoptotic pathway

Objetivo: o presente trabalho tem como objetivo avaliar o efeito da Benzidamina (BZD) na viabilidade celular em cultura de astrócitos e investigar o mecanismo de morte envolvido com seu efeito citotóxico. Métodos: para avaliar a viabilidade celular foi utilizado o ensaio de redução do brometo de 3-(4,5-Dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT), enquanto a citometria de fluxo foi utilizada para verificar o dano celular. O ensaio de imunofluorescência foi utilizado para verificar a expressão do marcador caspase-8, caspase9 e subunidade p65 do Fator nuclear kappa B (NFκB). A análise estatística para ensaio MTT e Citometria de Fluxo foi feita utilizando ANOVA com pós-teste de Dunett; Foi feito o teste t de Student para Imunofluorescência. A significância foi estabelecida em p < 0,05. Resultados: o ensaio de redução do MTT mostrou que o BZD (3,1 a 100 µg/mL) causou diminuição na viabilidade dos astrócitos. A citometria de fluxo mostrou que o efeito citotóxico do BZD foi causado pela ativação da via de morte apoptótica, evidenciada pela externalização da fosfatidilserina. A imunofluorescência revelou aumento na expressão de caspase-8 e nenhuma alteração na expressão de caspase-9, demonstrando que houve ativação da via extrínseca de apoptose. A concentração inibitória média (CI50) de BZD (26,13 µg/mL) também causou aumento na expressão de NFκB p65. Conclusão: em conjunto, os resultados do presente estudo sugerem que o BZD tem efeito citotóxico nas células astrocitárias, e esse efeito advém de sua capacidade de ativar a via apoptótica extrínseca.

The Effect of Shenqi Jianxin Formula （参芪健心方） on Cardiomyocyte Pyroptosis in Chronic Heart Failure Model Rats Based on the NLRP3/Caspase-1 Signaling Pathway / 中医杂志

ObjectiveTo investigate the possible mechanism of Shenqi Jianxin Formula （参芪健心方） in the treatment of chronic heart failure （CHF） from the perspective of pyroptosis. MethodsFifty-two rats were randomly divided into sham operation group （n=8） and modeling group （n=44）. In the modeling group， the anterior descending branch of the left coronary artery was ligated to construct CHF rat model. Forty successfully-modelled rats were randomly divided into model group， Entresto group， Shenqi Jianxin Formula group， MCC950 group and the combination group （Shenqi Jianxin Formula plus MCC950）， with 8 rats in each group. In Shenqi Jianxin Formula group， 7.4 g/（kg·d） of Shenqi Jianxin Formula was given by gavage， while in Entresto group， 68 mg/（kg·d） of Entresto suspension was given by gavage； in MCC950 group， MCC950 was injected intraperitoneally with 10 mg/kg once every other day， and in the combination group， 7.4 g/（kg·d） of Shenqi Jianxin Formula was given by gavage， and MCC950 was injected intraperitoneally with 10 mg/kg once every other day； 10 ml/（kg·d） of saline was given by gavage in the sham operation group and the model group. After 3 weeks of continuous intervention， serum brain B-type natriuretic peptide （BNP）， creatine kinase isoenzyme MB （CK-MB）， interleukin 1β （IL-1β）， and interleukin 18 （IL-18） levels were detected by ELISA； HE staining and MASSON staining were used to observe pathological changes in rat myocardium. Except for the Entresto group， western blot technique was used to detect the expression of NOD-like receptor protein 3 （NLRP3）， caspase-1， and apoptosis-associated speck-like protein possessing a caspase-recruiting domain （ASC）； RT-PCR was used to detect the expression of NLRP3 and caspase-1 mRNA. ResultsCompared with the sham operation group， HE staining of rats in the model group showed obvious myocardial injury， while MASSON staining showed increased area of collagen fibrosis， and serum BNP， CK-MB， IL-1β， IL-18， myocardial tissue NLRP3， caspase-1， ASC protein expression and NLRP3， caspase-1 mRNA expression were all elevated （P＜0.05）. Compared with those in the model group， cardiomyocyte injury of rats and collagen fibrosis area were reduced， and serum BNP， CK-MB， IL-1β， and IL-18 contents were all reduced in Shenqi Jianxin Formula group， Entresto group， MCC950 group， and the combination group； except for Entresto group， myocardial tissue NLRP3， caspase-1， ASC protein expression and NLRP3， caspase-1 mRNA expression were reduced in the remaining three medication group （P＜0.05）. Compared with Shenqi Jianxin Formula group， the MCC950 group and the combination group showed decreased serum IL-1β and IL-18 content， collagen fibrosis area， myocardial tissue NLPR3， caspase-1 protein expression， and caspase-1 mRNA expression， and decreased ASC and NLRP3 mRNA expression was shown in the combination group （P＜0.05）. Compared with MCC950 group， collagen fibrosis area was reduced， and serum IL-18 content， NLRP3， caspase-1 mRNA expression were reduced in the combination group （P＜0.05）. ConclusionShenqi Jianxin Formula can effectively improve the myocardial injury and heart failure in rats with CHF， and its mechanism may be related to the inhibition of cardiomyocyte pyroptosis through NLPR3/Caspase-1 pathway to reduce the level of intramyocardial inflammation. The combined use of MCC950 with Shenqi Jianxin Formula could more effectively inhibite myocardial pyroptosis， with better therapeutic result than single use of each part.

Molecular mechanism of adipose tissue inflammation induced by acute exposure to cooking oil fumes / 环境与职业医学

Background Cooking oil fumes are closely related to immune response, and adipose tissue also plays an important role in immune regulation. At present, the biological effect and mechanism of inflammation of adipose tissue induced by oil fume exposure are not clear yet. Objective To investigate the inflammatory effect of different exposure duration of cooking fumes on adipose tissue in mice and explore the role of Nod-like receptor pyrin domain 3 (NLRP3)/cysteinyl aspartate specific proteinase 1 (Caspase 1)/interleukin (IL)-1β signaling pathway. Methods Forty 8-week-old female C57BL/6J mice were randomly divided into 3-day control group (CON3 group), 7-day control group (CON7 group), 3-day oil fume exposure group (COF3 group), and 7-day oil fume exposure group (COF7 group), with 10 mice in each group. The mice were exposed to oil fumes in a cooking oil fume formation and exposure equipment (COFFEE) for 20 min, followed by a 10-min pause, 1 h a day for consecutive 3 d or 7 d. General condition of mice was observed and body weight was measured every day. After exposure, blood was sampled from the eyeball. Serum levels of IL-6, IL-27, and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The adipose tissue of mice was collected and observed after hematoxylin-eosin (HE) staining. The percentages of CD4+ and CD8+T cells in adipose tissue were detected by flow cytometry. Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of nuclear factor-κB (NF-κB), NLRP3, Caspase 1, and IL-1β in adipose tissue. Western blot was used to detect the expression levels of NLRP3, Caspase 1, and IL-1β in adipose. Results Compared with the corresponding control group, serum IL-6, IL-27, and IL-1β contents in the COF3 group and the COF7 group were significantly increased (P<0.05) except IL-6 in the COF3 group, and the levels in the COF7 group were significantly higher than those in the COF3 group (P<0.05). Vacuolar lipid droplets in adipocytes decreased, cytoplasm shrank, and inflammatory cells infiltrated in the COF7 group after HE staining. The flow cytometry results showed that the proportions of CD4+ and CD8+T cells in adipocytes of the COF3 group and the COF7 group were increased compared to the corresponding control group, with a significant increase in the COF7 group (P<0.05), and the CD4+/CD8+T ratio also significantly increased progressively in the two groups (P<0.05). The results of RT-qPCR showed that compared with the corresponding control group, the mRNA expression levels of NF-κB, NLRP3, Caspase 1, and IL-1β in adipose tissue of mice in the COF3 group and the COF7 group were significantly increased (P<0.05, P<0.01). The mRNA expression levels of mice in each exposure group gradually increased over time. The Western blot results showed that compared with the corresponding control group, the protein expressions of NLRP3 and Caspase 1 in the COF3 group were significantly increased (P<0.01), and the expression of IL-1β protein also increased but without statistical significance. The protein expressions of NLRP3, Caspase 1, and IL-1β in the COF7 group were significantly higher than those in the CON7 group (P<0.05, P<0.01). Conclusion Acute exposure to cooking oil fumes can induce significant inflammatory response in adipose tissue, and the effect gradually increases with the extension of exposure time. The mechanism of action may be related to the activation of NLRP3 inflammasome signaling pathway.

Effect of Modified Shenhong Tongluo Prescription on Cell Apoptosis in Rats with Myocardial Ischemia-reperfusion Injury by Bcl-2/Bax/Caspase-3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of modified Shenhong Tongluo prescription on cell apoptosis in rats with myocardial ischemia-reperfusion injury （MIRI）. MethodSixty Sprague-Dawley （SD） rats were randomly divided into a blank group， a model group， low-， medium-， and high-dose groups of modified Shenhong Tongluo prescription， and a simvastatin group. Except for the blank group， a rat model of MIRI was prepared by ligating the left anterior descending coronary artery. Starting from the first day after successful modeling， the blank group （1.0 mL·kg-1 physiological saline）， model group （1.0 mL·kg-1 physiological saline）， low-， medium-， and high-dose groups of modified Shenhong Tongluo prescription （1.031， 2.063， and 4.126 g·kg-1 Shenhong Tongluo prescriptiona standard concentrate）， and simvastatin group （0.71 mg·kg-1 simvastatin） were orally administered once daily for 2 weeks. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of cardiomyocytes. Enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of serum creatine kinase isoenzyme （CK-MB）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α）. TdT-mediated dUTP nick-end labeling（TUNEL） staining was used to detect the apoptosis rate of rat cardiomyocytes. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and caspase-3. ResultCompared with the blank group， in the model group， HE staining showed disturbed arrangement of cardiomyocytes， incomplete fibers， focal necrosis of cardiomyocytes， and inflammatory cell infiltration； serum CK-MB， IL-6， and TNF-α levels were significantly increased （P<0.05）； apoptosis rate of cardiomyocytes was significantly increased （P<0.01）， with significantly increased expression levels of Bax and Caspase-3 proteins， and significantly decreased Bcl-2 expression （P<0.05）. Compared with the model group， the low-， medium-， and high-dose groups of modified Shenhong Tongluo prescription significantly reduced CK-MB， IL-6， and TNF-α levels （P<0.05）， significantly downregulated cardiomyocyte apoptosis rate （P<0.05）， significantly decreased Bax and Caspase-3 proteins， and significantly increased Bcl-2 expression levels （P<0.01）. In the modified Shenhong Tongluo prescription groups， the expression levels of Bax and Caspase-3 proteins significantly decreased with increasing dosage， while the expression level of Bcl-2 significantly increased with increasing dosage of modified Shenhong Tongluo prescription （P<0.05）. ConclusionShenhong Tongluo prescription can alleviate myocardial tissue pathological damage and reduce myocardial cell apoptosis， possibly by inhibiting Caspase-3 and Bax expression and promoting Bcl-2 expression.

Mechanism of Buzhong Yiqitang Intervening in Pyroptosis of AIT in NOD.H-2h4 Mice Based on NLRP3/Caspase-1/GSDMD Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Buzhong Yiqitang on pyroptosis in autoimmune thyroiditis （AIT） mice based on the NOD-like receptor hot protein domain related protein 3 （NLRP3）/cysteinyl aspartate specific proteinase-1（Caspase-1）/Gasdermin D （GSDMD） pathway. MethodSixty NOD.H-2h4 mice were divided into normal group， model group， low， medium， and high dose groups （4.10， 8.19， 16.38 g·kg-1）of Buzhong Yiqitang， and selenium yeast tablet group （0.26 mg·kg-1）， with 10 mice in each group. Except for the normal group， all other groups were given 0.05% NaI by gavage for eight weeks to establish a model and then received the drug treatment for eight weeks. The serum levels of thyroid peroxidase antibody （TPO-Ab） and thyroglobulin antibody （TgAb） were detected using enzyme-linked immunosorbent assay （ELISA） method. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in mouse thyroid tissue. The immunohistochemical method was used to detect the protein expression of NLRP3， Caspase-1， interleukin-1β （IL-1β）， and interleukin-18 （IL-18）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of NLRP3， Caspase-1， IL-1β， and IL-18. Western blot was used to detect the levels of pyroptosis-related proteins in thyroid tissue. ResultCompared with the normal group， the serum levels of TPO-Ab and TgAb in the model group were significantly increased （P<0.01）. Thyroid follicles either increased in a cubic shape or were damaged and atrophied， with a large number of lymphocytes infiltrating around the follicles. Compared with the model group， the levels of TPO-Ab and TgAb in other groups were significantly reduced （P<0.01）， and the morphology and structure of follicles were improved. The degree of lymphocyte infiltration was reduced. Among them， the medium dose group of Buzhong Yiqitang had the most significant reduction and improvement effect. Compared with the normal group， the positive products and mRNA expression of NLRP3， Caspase-1， IL-1β， and IL-18 proteins in the thyroid tissue of the model group significantly increased （P<0.01）， and the protein expression levels of NLRP3， cleaved Caspase-1， IL-1β， IL-18， and GSDMD-N were significantly increased （P<0.05， P<0.01）. Compared with the model group， the positive products and mRNA expression of NLRP3， Caspase-1， IL-1β， and IL-18 proteins in other groups were significantly reduced （P<0.05， P<0.01）， with the most significant reduction effect in the medium dose group of Buzhong Yiqitang. The protein expression levels of NLRP3， cleaved Caspase-1， IL-1β， IL-18， and GSDMD-N were significantly reduced （P<0.05， P<0.01）. ConclusionBuzhong Yiqitang can improve AIT， and its mechanism may be achieved by regulating the NLRP3/Caspase-1/GSDMD signaling pathway to inhibit pyroptosis.

Role of HK2 and VDAC1 in Diacetylmorphine-induced Cardiomyocyte Apoptosis / 昆明医科大学学报

Objective To investigate the role of HK2 and VDAC1 in diacetylmorphine-induced cardiomyocyte apoptosis.Methods A dose-escalation method was used to establish a rat model of diacetylmorphine addiction.Forty SD rats were randomly divided into three groups,the normal group(n=10)was injected with an equal amount of saline subcutaneously,the model group(n=15)was injected with 5 mg/kg of diacetylmorphine for the first time,and then the dose was increased by 2.5 mg/(kg·d)day by day for 20 days,and the group of model +10 D(n=15)continued to increase the dose based on the model group up to the 10th day.Lactate dehydrogenase(LDH)and glutamic oxaloacetic transaminase(GOT)were detected by ELISA;HE staining was used to observe the pathological changes of myocardial tissues in each group;TUNEL staining was used to detect apoptosis in myocardial tissues in each group;and immunohistochemistry,RT-q-analysis,and immunochemistry were used to detect apoptosis in myocardial tissues in each group.Immunohistochemistry,RT-qPCR and Western bl-ot were used to detect the mRNA and protein expression of HK2,VDAC1 and apoptosis-related factors.Results HE staining revealed that myocardial tissues exhibited different degrees of damage with the prolongation of diacetylmorphine intervention.Compared with the normal group,serum LDH,GOT content and myocardial apoptosis rate increased in the model group,mRNA and protein levels of HK2 and anti-apoptotic factor Bcl-2 decreased,mRNA and protein levels of VDAC1 and pro-apoptotic factors Bax and Caspase-3 increased,and the protein level of Clevead Caspase-3 increased;in the model +10 D group the above indexes,there was a statistically significant difference(P<0.05).Conclusion Diacetylmorphine can cause cardiomyocyte apoptosis,and VDAC1 may be involved in the process of cardiomyocyte apoptosis caused by diacetylmorphine.

Impact of andrographolide on intestinal inflammation in rats with diarrhea type irritable bowel syndrome by regulating the NLRP3/caspase-1 signaling pathway / 中华内分泌外科杂志

Objective:To investigate the impact of andrographolide (AND) on intestinal inflammation in rats with diarrhea type irritable bowel syndrome (IBS-D) and its regulatory mechanism on the NLRP3/caspase-1 signaling pathway.Methods:After the IBS-D rat model was established by low concentration acetic acid combined with restraint stress, the rats were grouped into control group, model group, positive drug group, AND low-dose (AND-L) group, AND medium dose (AND-M) group, and AND high-dose (AND-H) group, with 10 rats in each group. The score of fecal traits and fecal water content of rats in each group were detected; the abdominal withdrawal reflex (AWR) of rats in each group was scored; HE staining was applied to observe the changes of colonic histopathology of rats in each group; enzyme linked immunosorbent assay (ELISA) was applied to detect the levels of tumor necrosis factor-α (TNF- α), interleukin-6 (IL-6), interleukin-1 β (IL-1 β), and interleukin-18 (IL-18) in the colon tissue of rats in each group; Western blot was applied to detect the expression levels of NLRP3, ASC, and caspase-1 proteins in the colon tissue of rats in each group. Results:The scores of fecal traits of rats in the control group was 2.43±0.19, fecal water content was 31.76±2.81, AWR score was 0.43±0.02, TNF-α was 123.49±12.35, IL-6 was 76.45±6.23, IL-1 β was 195.76±15.14 and IL-18 was 167.31±13.92, the protein expression levels of NLRP3 was 0.96±0.06, ASC was 1.01±0.08, and caspase-1 was 0.94±0.06. The scores of fecal traits in model group was 6.12±0.58, fecal water content was 65.24±4.13, AWR score was 2.42±0.18, which were higher than those in control group ( P<0.05), the TNF- α in model group was 315.73±19.47, IL-6 was 231.97±14.65, IL-1 β was 435.83±28.67, IL-18 was 382.56±26.84, the protein expression levels of NLRP3 was 2.41±0.18, ASC was 2.23±0.15, and caspase-1 was 2.15±0.16, which were higher than those in control group ( P<0.05). The scores of fecal traits in the low, medium, and high dose AND groups were 5.38±0.46, 4.57±0.38, 3.31±0.27, fecal water content were 54.68±3.67, 46.87±3.75, 38.11±3.10, AWR scores were 1.79±0.16, 1.35±0.10, 0.69±0.04, which were higher than those in model group ( P<0.05), the TNF- α in the low, medium, and high dose AND groups were 268.65±17.23, 224.91±16.36, 178.16±14.65, IL-6 were 187.74±14.57, 159.64±11.39, 124.18±8.62, IL-1 β were 369.51±21.96, 314.72±23.64, 263.93±16.82, IL-18 were 334.72±25.17, 280.16±21.43, 235.67±19.32, the protein expression levels of NLRP3 were 1.94±0.15, 1.56±0.12, 1.25±0.09, ASC were 1.89±0.14, 1.61±0.13, 1.28±0.10, and caspase-1 were 1.76±0.14, 1.49±0.11, 1.20±0.09, which were higher than those in model group ( P<0.05) . Conclusion:AND may alleviate intestinal inflammation in IBS-D rats by inhibiting the NLRP3/caspase-1 signaling pathway.

Exercise-induced chronic fatigue impairs working memory in rats via basal ganglia output nuclei / 神经解剖学杂志

Objective:To investigate possible neuromodulatory mechanisms involved in the involvement of parvalbu-min(PV)expression in the basal ganglia output nuclei,entopeduncular nucleus(EPN)and substantia nigra pars etic-ulata(SNr),in exercise-induced chronic fatigue impairs working memory capacity.Methods:Male SD rats were divid-ed into control group and Fatigue group by random number method,and a three-stage incremental load treadmill training program was selected to establish a chronic exhaustion exercise-induced fatigue rat model.The working memory ability of rats was assessed by the Y-maze autonomous alternation experiment.Immunohistochemical staining was used to ob-serve the expression of parvalbumin(PV)positive neurons and cysteine aspartate-specific protease-3(caspase-3)in EPN and SNr of rats.Results:The accuracy of voluntary alternation in the fatigue group was obviously lower than that in control group(P＜0.05).The results of immunohistochemical staining showed that the density of PV positive neu-rons and the degree of positive fiber staining in EPN and SNr in the fatigue group were obviously lower than those in the control group(P＜0.05,P＜0.01).The number of caspase-3 positive cells per unit area of EPN and SNr in the fa-tigue group was obviously higher than that in the control group(P＜0.05,P＜0.01).Conclusion:The mechanism of impairing working memory in rats caused by exercise-induced chronic fatigue may be related to the apoptosis of PV posi-tive neurons in EPN and SNr.

The mechanism of target regulating of miR-421 to Menin/Caspase-3 pathway for depression / 实用医学杂志

Objective To explore the mechanism of miR-421 affecting the occurrence and development of depression.Methods A depressive rat model was established by single intraperitoneal injection of lipopolysaccharide(LPS),and depressive behavior was detected by glucose preference test and open-field test.miRNA microarray chips and RT-PCR were used to analyze the expression level of miR-421 in hippocampus of the depressed rats.TargetScan database and mi RDB database were used to predict the target genes of miR-421.Dual luciferase reporter gene assay was used to observe the binding of miR-421 to the target genes.The impact of over-expression and inhibition of miR-421 on target genes was observed,then the influence of over-expression and inhibition of target genes on downstream factors was observed,and the related mechanism of miR-421 on depression was explored.Results miRNA microarray chips and RT-PCR assay showed that miR-421 was highly expressed in the hippocampus of the depressed rats(P<0.001),Inhibition of miR-421 expression could significantly restore the body weight and exercise ability of the depressed rats(P<0.001).Binding targets of Menin and miR-421 were predicted by TargetScan database,and interaction between Menin and miR-421 was demonstrated by dual-luciferase reporter gene assay.Menin expression was down-regulated while miR-421 was overexpressed(P<0.001),whereas it was up-regulated as miR-421 was inhibited(P<0.001).qPCR indicated that expressions of Caspase-3 and NF-κB in the hippocampus of the depressed rats was significantly increased(P<0.001),and IL-1β expression in the hippo-campus was significantly increased(P<0.01).When the expression of Menin was inhibited,the expressions of Caspase-3,NF-κB and IL-1β were increased(P<0.001),while the expressions of Caspase-3,NF-κB and IL-1β were decreased when Menin was overexpressed(P<0.001).Conclusions Inhibition of miR-421 expression can increase Menin expression,decrease Caspase-3 content,and reduce neuroinflammatory response,thereby improving depressive symptoms.

Regulatory mechanisms and therapeutic strategies for pyroptosis after spinal cord injury / 中国组织工程研究

BACKGROUND:Cell death and neuroinflammation are two important targets in the treatment of spinal cord injury.Pyroptosis is a programmed cell death closely related to neuroinflammation and targeted inhibition of pyroptosis after spinal cord injury is a promising therapeutic strategy. OBJECTIVE:To summarize the molecular mechanism,positive and negative regulatory factors and therapeutic strategies of pyroptosis in spinal cord injury. METHODS:The search terms were"spinal cord injury,pyroptosis,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),Caspase,Gasdermin D(GSDMD),IL-1β,IL-18"and 93 English literatures included in PubMed and Web of Science were finally selected for review. RESULTS AND CONCLUSION:As a newly discovered programmed cell death,pyroptosis has been shown to play an important role in the secondary injury stage after spinal cord injury.Among the regulatory factors of pyroptosis after spinal cord injury,CD73,NRF2,GDF-11,dopamine,FANCC and miR-423-5P could inhibit pyroptosis,while TLR4 and Aopps could promote pyroptosis.In terms of treatment,the active ingredients of traditional Chinese medicine(paeonol,tripterine,betulinic acid,piperine,kaempferol,and camptothecin),exosomes of various cell origins,and some drugs(metformin,topotecan,lithium,zinc,and carbon monoxide-releasing molecule 3)can effectively inhibit pyroptosis and reduce secondary spinal cord injury,but the toxicity and specific dose of these drugs need to be further studied.The specific molecular mechanism by which pyroptosis aggravates spinal cord injury is still poorly understood.The role of non-classical pathways and other inflammasomes is worth further exploration.At present,the research on pyroptosis after spinal cord injury only stays at the animal experiment stage.There are no related clinical studies and no approved targeted therapeutic drugs.(6)The application of pyroptosis after spinal cord injury has great potential,and its specific regulatory mechanism should be further studied in the future to provide a new target for the treatment of spinal cord injury.

Grape seed extract inhibits apoptosis of rat growth plate chondrocytes and promotes tibial bone growth / 中国组织工程研究

BACKGROUND:Grape seed extract can inhibit chondrocyte apoptosis and aging,and improve osteoarthritis.However,the effects of grape seed extract on the apoptosis of chondrocytes in the growth plate and tibial growth are still unclear. OBJECTIVE:To investigate the effect of grape seed extract on interleukin-1β-induced apoptosis in rat growth plate cells and on tibial bone growth. METHODS:(1)Cell experiment:Growth plate chondrocytes from Sprague-Dawley rats were isolated,cultured,and identified.The cells were then randomly divided into control group,model group,grape seed extract group,miR-138-5p NC group and miR-138-5p inhibitor group.In the model group,20 ng/mL interleukin-1β was used to induce apoptosis in rat growth plate chondrocytes.In the grape seed extract group,20 ng/mL interleukin 1β was added along with 10 μmol/L grape seed extract solution for 48 hours.Cells in the miR-138-5p NC and inhibitor groups were transfected with 5 nmol/L miR-138-5p NC and 5 nmol/L miR-138-5p inhibitor for 12 hours,respectively,followed by addition of 20 ng/mL interleukin-1β.qRT-PCR was used to detect miR-138-5p and caspase-3 expression.Luciferase reporter assay was used to analyze the relationship between miR-138-5p and caspase-3 targeting.Cell counting kit-8 was used to detect cell proliferation activity.Flow cytometry was used to detect cell apoptosis.Caspase-3 and Bcl-2 protein expressions were detected by western blot.(2)Animal experiment:The animals were divided into normal control group,grape seed extract group and miR-138-5p inhibitor group.The effects of grape seed extract on epiphyseal closure and tibial growth of the tibial plateau in rats were observed. RESULTS AND CONCLUSION:miR-138-5p had a targeting relationship with caspase-3.Compared with the control group,cell proliferation was significantly reduced,apoptosis was significantly increased(P＜0.01),miR-138-5p,Bcl-2 expression was reduced(P＜0.01),and caspase-3 expression was increased(P＜0.01)in the model group.Compared with the mod group,the grape seed extract group showed a significant increase in cell proliferation,a significant decrease in apoptosis(P＜0.01),an increase in miR-138-5p and Bcl-2 expression(P＜0.01)and a decrease in caspase-3 expression(P＜0.01).Compared with the grape seed extract group,the miR-138-5p inhibitor group showed a significant decrease in cell proliferation,a significant increase in apoptosis(P＜0.01),a decrease in miR-138-5p and Bcl-2 expression(P＜0.05 or P＜0.01),and an increase in caspase-3 expression(P＜0.05).Intragastric administration of grape seed extract could delay epiphyseal closure of the tibial plateau and promote tibial bone growth in rats,whereas miR-138-5p inhibitor intervention inhibited the effect of grape seed extract on tibial bone growth in rats.To conclude,grape seed extract can inhibit apoptosis of rat growth plate chondrocytes through regulating the miR-138-5p/caspase-3 pathway,improve epiphyseal closure of rat tibial plateau and promote tibial bone growth.

Effects of aerobic exercise on the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice / 中国组织工程研究

BACKGROUND:β-amyloid protein and Tau protein have adverse effects on the cognitive function of Alzheimer's disease patients,and Notch1 and Caspase-3 can regulate the expression of β-amyloid protein and Tau protein.It is not clear whether Notch1 and Caspase-3 mediate the process of aerobic exercise to improve the cognitive ability of Alzheimer's disease patients.At present,there is a lack of studies on the effect of long-term aerobic exercise on the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice. OBJECTIVE:To observe the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice undergoing long-term aerobic exercise and to investigate the effects of Notch1 and Caspase-3 in Alzheimer's disease mice. METHODS:Wild type and APP/PS1 double-transgenic Alzheimer's disease mice aged 3 months were randomly divided into four groups:wild control group,wild exercise group,Alzheimer's disease control group and Alzheimer's disease exercise group,with 20 mice in each group.Mice in the control groups were not subjected to exercise,while those in the exercise groups received aerobic exercise intervention for 5 months.After the exercise intervention,Morris water maze was used to detect the spatial learning and memory ability of mice.Real-time PCR,immunofluorescence and western blot were used to detect the expressions of Aβ1-42,Tau,Notch1 and Caspase-3 in the hippocampal tissues of mice in each group. RESULTS AND CONCLUSION:The spatial learning and memory ability of Alzheimer's mice was significantly worse than that of wild-type mice(P＜0.05).The spatial learning and memory ability of mice in the exercise groups were significantly better than that in the corresponding control groups(P＜0.05).The expressions of Aβ1-42,Tau,Notch1 and Caspase-3 in the hippocampus were significantly higher in the Alzheimer's disease control group than the wild control group(P＜0.05)and were significantly lower in the Alzheimer's disease exercise group than the Alzheimer's disease control group(P＜0.05).To conclude,long-term aerobic exercise can improve the spatial learning and memory ability of Alzheimer's disease mice,which may be related to the decreased expression of Notch1,Caspase-3,Aβ1-42 and Tau protein in the hippocampus of Alzheimer's disease mice.

Moxibustion inhibits NLRP3/Caspase-1 pathway mediated cell pyroptosis and alleviates cerebral ischemia/reperfusion injury / 中国组织工程研究

BACKGROUND:It was found that moxibustion can inhibit the inflammatory factors in the serum of rats with cerebral ischemia/reperfusion injury,resist oxidative stress,inhibit cell apoptosis,and effectively reduce cerebral ischemia/reperfusion injury. OBJECTIVE:To observe the effects of different moxibustion intervention time on the expression levels of nucleotide binding oligomerization domain-like protein 3 inflammasome(NLRP3),cysteine aspartase(caspase-1),apoptosis-related speck-like protein,exfoliatin-D protein,interleukin-1β and interleukin-18 in rats with cerebral ischemia/reperfusion injury,and to explore its action mechanism. METHODS:SD rats were randomly divided into sham operation group(n=9)and operation group(n=36).The model of focal cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion in the operation group.After successful modeling,the rats in the operation group were further divided into model group,moxibustion 10-minute group,moxibustion 15-minute group and moxibustion 30-minute group,with 9 rats in each group.Rats in the moxibustion 10-minute,15-minute and 30-minute groups were given moxibustion at"Baihui,Dazhui and Zusanli",respectively,once a day for a total of 7 days.The neurological deficits of rats were evaluated by LONGA method.The cerebral infarction was observed by 2,3,5-triphenyltetrazolium chloride staining.The pathological changes of brain tissue were observed by hematoxylin-eosin staining.The contents of interleukin-1β and interleukin-18 in serum of rats in each group were detected by ELISA.Immunohistochemistry and western blot assay were used to detect the expression levels of NLRP3,caspase-1,apoptosis-related spot-like protein and gasdermin D in the ischemic cortex of rats in each group. RESULTS AND CONCLUSION:Compared with the sham operation group,the neurological deficit score of the model group was significantly increased(P<0.01).Compared with the model group,the neurological deficit score of the moxibustion groups was significantly reduced(P<0.01).Compared with the sham operation group,the infarct volume of the model group was significantly increased(P<0.01).Compared with the model group,the infarct volume of the moxibustion groups was significantly reduced(P<0.01);the infarct volume of the rats was smallest in the moxibustion 30-minute group(P<0.05).Compared with the model group,the contents of inflammatory factors interleukin-1β and interleukin-18 in the serum of rats in the moxibustion groups were decreased(P<0.01).Compared with the moxibustion 10-minute group,the contents of inflammatory factors in the serum of rats in the moxibustion 30-minute group were significantly decreased(P<0.05).Compared with the model group,the expression of NLRP3,apoptosis-related spot-like protein,Caspase-1 and gasdermin D protein in the ischemic cortex of the moxibustion groups was significantly decreased(P<0.01).Compared with the moxibustion 10-minute and 15-minute groups,the expression of protein in the moxibustion 30-minute group was significantly decreased(P<0.05).It is concluded that moxibustion at Baihui,Dazhui and Zusanli can reduce cerebral ischemia/reperfusion injury,among which moxibustion for 30 minutes has the best effect,and its mechanism may be related to the inhibition of pyroptosis mediated by NLRP3/Caspase-1 pathway.

Effects of Electroacupuncture on Cyt-C and Caspase-9 Expression in Hippocampal Tissue of Rats with Chronic Pain and Depression Comorbidity / 中国中医药信息杂志

Objective To observe the effects of electroacupuncture(EA)at"Hegu"and"Taichong"on the expressions of cytochrome C(Cyt-C)and Caspase-9 in the hippocampus of rats with chronic pain and depression comorbidity(CPDC);To explore its potential mechanism for the treatment of CPDC.Methods A total of 60 SD rats were randomly divided into sham-operation group,model group,medication group and EA group,with 15 rats in each group.The CPDC model was induced by twice injection of complete Freund's adjuvant into the plantar of the left hind paw.EA group was applied to bilateral"Hegu"and"Taichong"for 20 min,the medication group were treated with duloxetine suspension 10 mg/kg by gavage for 14 days.The mechanical paw withdrawal threshold and thermal withdrawal latency were measured on day 7,14,21 and 28 after the first injection,tail suspension test and sucrose preference test were performed on day 14 and 28,Nissl staining was used to observe hippocampal neurons and the number of Nissl body,the protein expressions of Cyt-C and Caspase-9 in hippocampal tissue were detected by Western blot and immunohistochemistry.Results Compared with the sham-operation group,the mechanical paw withdrawal threshold and thermal withdrawal latency of the model group significantly decreased(P<0.01),the tail suspension immobility time increased(P<0.01),the percentage of sucrose preference decreased(P<0.01),the neurons were thinly arranged,the neurons were damaged and lost,and the number of Nissl body were less(P<0.01),the apoptotic rate of hippocampal neurons increased(P<0.01),the expression of Cyt-C,Caspase-9 and cleaved-Caspase-9/Caspase-9 ratio in hippocampal tissue increased(P<0.01).Compared with the model group,the mechanical paw withdrawal threshold and thermal withdrawal latency of the EA group and medication group significantly increased(P<0.01),the tail suspension immobility time decreased(P<0.01),the percentage of sucrose preference increased(P<0.01),the loss of neurons in hippocampus was reduced,the cells were arranged neatly,and the nucleoli were clear,the number of Nissl body increased(P<0.01),the apoptotic rate of hippocampal neurons decreased(P<0.01),and the expression of Cyt-C,Caspase-9 and cleaved-Caspase-9/Caspase-9 ratio in hippocampal tissue decreased(P<0.01).Conclusion EA at"Hegu"and"Taichong"can alleviate pain and depression in CPDC rats,which may be related to inhibiting the expressions of Cyt-C and Caspase-9 in hippocampal tissue and inhibiting the apoptosis of hippocampal neurons,thus playing a neuroprotective effect.

Effects of sleep disorders on hematopoietic stem cells in bone marrow of irradiated mice / 中华放射医学与防护杂志

Objective:To investigate the effects of sleep disorders (SD) on the radiation injury of hematopoietic stem cells (HSCs) in bone marrow (BM).Methods:Totolly 56 C57BL/6J male mice aged 6-8 weeks were enrolled in this study. They were subjected to whole body irradiation of 60Co γ-rays with doses of 5.0 and 7.5 Gy. A SD model was established using a SD device. According to the random number table method, the mice were divided into seven groups: the control group (Con group), the SD group, the mere radiation group (IR group), the group of post-irradiation SD (IR+ SD group), the group of post-irradiation SD treated with phosphate buffer solution (IR+ SD+ PBS group), the group of post-irradiation SD treated with GSK2795039 (IR+ SD+ GSK group), and the group of post-irradiation SD treated with N-acetylcysteine (IR+ SD+ NAC group), with in eight mice each group. The changes in the peripheral blood of the mice after 5.0 Gy irradiation were detected using the collected tail venous blood, and the survival rates of the mice after 7.5 Gy irradiation were observed. The changes in the density and count of bone marrow cells were observed using hematoxylin and eosin (HE) staining. The number of hematopoietic stem cells in bone marrow (LSK cells), as well as their apoptosis level and changes in cell cycle, were detected using flow cytometry. Furthermore, indicators of LSK, such as reactive oxygen species(ROS) and mitochondrial-derived reactive oxygen species (mtROS), were analyzed. Nicotinamide adenine dinucleotide phosphate (NADP+ /NADPH) and glutathione (GSSG/GSH) were detected using an enzyme microplate reader in order to observe the oxidative stress level of LSK. Furthermore, flow cytometry was employed to sort the LSK cells from the mice, and flow cytometry was used to detect the expression of NADPH oxidase 2(NOX2) and cysteinyl aspartate specific proteinnase-1(Caspase-1), and polymerase chain reaction (PCR) was used to detect the expression of inflammatory factors such as NOX1-4, interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 18 (IL-18), and tumor necrosis factor α (TNF-α). Results:Compared to the IR group, the IR+ SD group exhibited significantly slower recovery of white blood cells (WBC) and platelets (PLT) ( t = 4.39, 6.37, P < 0.05), the bone marrow cell count decreasing from (2.14 ± 0.38) × 10 7 to (3.59 ± 0.29) × 10 7 ( t = 8.55, P < 0.05), significantly decreased proportion of G 0-phase LSK cells, significantly increased proportion of apoptotic cells ( t = 7.53, 8.21, P < 0.05), and significantly increased DCFH-DA, MitoSOX, and NADP+ /NADPH ( t = 22.99, 29.47, 3.77, P<0.05). In the case of IR, SD further promoted the activation of NOX2 and led to increases in the mRNA expression of downstream inflammatory factors such as IL-1β, IL-6, IL-18, and TNF-α ( t = 6.95, 6.01, 8.39, 4.91, 5.56, P < 0.05). Inhibition of NOX2-ROS could prevent the SD-induced aggravation of post-irradiation hematopoietic injury. This significantly reduced the apoptotic rate of LSK cells and the expression of inflammatory factors, ultimately accelerating the hematopoietic recovery of LSK cells ( t = 9.24, 3.92, P < 0.05). Conclusions:SD can aggravate the IR-induced injury of hematopoietic stem cells in bone marrow, primarily by activating the NOX2-ROS-Caspase-1 axis. This will increase the levels of intracellular inflammatory factors and ROS, promote cell apoptosis, and ultimately inhibit the hematopoietic recovery of bone marrow.

Mechanism Study on Chaihuang Qingyi Huoxue Granules Reduce Inflammatory Damage in Severe Acute Pancreatitis Rats by Suppressing NLRP3 Inflammasome Activation / 中药新药与临床药理

Objective To investigate the protective effect and mechanism of Chaihuang Qingyi Huoxue Granules on pancreatic tissue of rats with severe acute pancreatitis,and to observe its regulation on NLRP3 inflammasome activation.Methods Sixty-four SD rats were randomly divided into sham-surgery(SO)group,severe acute pancreatitis model(SAP)group,Chaihuang Qingyi Huoxue Granules(CH)group,and MCC950(NLRP3 inhibitor)group.Each group was further divided into 12-hour and 24-hour subgroups,with rats in each group.The SAP group,CH group,and MCC950 group were retrogradely injected with 3.5%sodium taurocholate(2 mL·kg-1)into the pancreatic ducts to establish SAP model.The MCC950 group was immediately intraperitoneally injected with MCC950(1 mg·mL-1)after model preparation.After awakening from anesthesia,the CH group was administrated by gavage with Chaihuang Qingyi Huoxue Granules solution(0.35 g·mL-1)once every 6 hours.Ascites,abdominal aortic blood,and pancreatic tissue were collected at 12 hours and 24 hours after SAP model construction.The serum amylase and lipase activities were detected using an automated biochemical analyzer.HE staining was used to observe pancreatic injury.Serum levels of IL-18 and IL-1β were detected by ELISA.The expressions of gene and proteins related to the activation of NLRP3 inflammasome were analyzed by IHC,qRT-PCR and Western Blot.Results Compared with the SAP group,the pathological damage of pancreatic tissues in the CH and MCC950 groups was significantly reduced,and the pathological score was significantly reduced(P＜0.05).The levels of serum lipase,amylase,IL-18,and IL-1β were also significantly decreased(P＜0.05).After treatment with Chaihuang Qingyi Huoxue Granules or intraperitoneal injection of NLRP3 inhibitor,the positive expressions of NLRP3,ASC and Caspase-1 in pancreatic tissues,as well as the mRNA levels of NLRP3,ASC and Caspase-1,the protein levels of NLRP3,ASC,Pro-Caspase-1 and Caspase-1 were significantly reduced compared to the SAP group(P＜0.05).Conclusion Chaihuang Qingyi Huoxue Granules can inhibit the activation of NLRP3 inflammasome,reduce the mRNA and protein expressions of NLRP3,ASC and Caspase-1 in pancreatic tissues,and suppress the release of the downstream inflammatory factors IL-18 and IL-1β and alleviate pancreatitis damage in SAP model rats.

Effects of Ziziphi Spinosae Semen-Albiziae Flos on the IRE1α/ASK1/JNK Pathway of Endoplasmic Reticulum Stress in Hippocampus of Depression Model Rats / 中药新药与临床药理

Objective Based on the endoplasmic reticulum stress(ERS)inositol-requiring enzyme-1α(IRE1α)/apoptosis signal-regulated kinase 1(ASK1)/c-Jun N-terminal kinase(JNK)pathway to investigate the intervention effect of Ziziphi Spinosae Semen(ZSS)-Albiziae Flos(AF)on the depression model of rats,which were prepared by solitary rearing combined with chronic unpredictable mild stress(CUMS).Methods A total of 144 rats were randomly divided into normal group,model group,high-,medium-and low-dose groups of ZSS-AF,and Venlafaxine group.In addition to the normal group,the rats in other groups were isolated for 21 days combined with CUMS to prepare the depression model.The behavioral changes of rats were observed by open field test and Morris water maze test.The ultrastructural changes of hippocampal neurons were observed by transmission electron microscope.TUNEL staining was used to observe the apoptosis of nerve cells in hippocampus.The protein expression levels of IRE1α,phosphorylated(P)-IRE1α,ASK1,P-ASK1,JNK,P-JNK,B cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and cysteme aspartate specific protease-3(Caspase-3)in hippocampus were detected by Western Blot.The mRNA expression levels of IRE1α,ASK1,JNK,Bax,Bcl-2 and Caspase-3 in hippocampus were detected by Real-Time PCR.Results Compared with the normal group,the scores of horizontal movement and vertical movement in the open field test of rats in the model group were decreased(P＜0.01).In the water maze test,the escape latency was increased and the number of crossing the original platform was decreased(P＜0.01).The number of hippocampal apoptosis was increased(P＜0.01).The protein expression levels of P-IRE1 α/IRE1 α,P-ASK1/ASK1,P-JNK/JNK,Bax,Caspase-3 and mRNA expressions of IRE1α,ASK1,JNK,Bax,Caspase-3 in hippocampus were increased,while the protein and mRNA expression levels of Bcl-2 were decreased(P＜0.01).Compared with model group,the scores of horizontal movement and vertical movement in the open field test of rats in each dose ZSS-AF groups and Venlafaxine group were increased(P＜0.01).In the water maze test,the escape latency was decreased and the times of crossing the original platform was increased(P＜0.01).The number of hippocampal apoptosis was decreased(P＜0.01).The mRNA expression levels of P-IRE1α/IRE1α,P-ASK1/ASK1,P-JNK/JNK,Bax,Caspase-3 protein and IRE1α,ASK1,JNK,Bax,Caspase-3 in hippocampus were decreased,while the protein and mRNA expression levels of Bcl-2 were increased(P＜0.05,P＜0.01).The effect of medium-dose ZSS-AF group was better than that of high-and low-dose groups.Conclusion ZSS-AF may play an antidepressant role by regulating IRE1α/ASK1/JNK pathway of endoplasmic reticulum stress.

Hei Xiaoyaosan Regulates Fas/FasL/Caspase-8/Caspase-3 Signaling Pathway to Inhibit Neuronal Apoptosis in AD Rats / 中国实验方剂学杂志

ObjectiveTo explore the effect and mechanism of Hei Xiaoyaosan in regulating the tumor necrosis factor receptor superfamily member 6 （Fas）/Fas ligand （FasL）/cysteine protease-8 （Caspase-8）/cysteine protease-3 （Caspase-3） signaling pathway to intervene in neuronal apoptosis and prevent Alzheimer's disease （AD）. MethodNinety SPF-grade SD male rats of 4 months old were selected and randomly grouped as follows： 10 rats in the blank group， 10 rats in the sham group （bilateral hippocampus injected with 1 μL normal saline）， and 70 rats in the modeling group ［bilater hippocampus injected with 1 μL amyloid-beta protein 1-42 （Aβ1-42） solution for the modeling of AD］. Fifty successfully modeled rats were selected and randomly assigned into model， donepezil hydrochloride （0.45 mg·kg-1）， and high-， medium-， and low-dose （15.30， 7.65， 3.82 g·kg-1） Hei Xiaoyaosan groups. Rats were administrated with corresponding agents by gavage once a day for 42 days. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling （TUNEL） was employed to observe the apoptosis of neurons in the cortex and hippocampus， and immunohistochemistry （IHC） was used to detect the expression of B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax） in the hippocampus. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of Fas， FasL， and Fas-associated protein with death domain （Fadd）. Western blot was used to determine the protein levels of Fas， FasL， Fadd， Caspase-3， cleved Caspase-3， Caspase-8， and cleved Caspase-8. ResultCompared with the blank group and sham group， the model group showed increased apoptosis rate in the cortex and hippocampus （P<0.01）， elevated Bax level （P<0.01）， lowered Bcl-2 level （P<0.01）， up-regulated mRNA levels of Fas， FasL， and Fadd in the hippocampus （P<0.01）， and up-regulated protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.01）. Compared with the model group， donepezil hydrochloride and Hei Xiaoyaosan at high and medium doses decreased the apoptosis rate in the cortex and hippocampus （P<0.05， P<0.01）， lowered the Bax level （P<0.01）， elevated the Bcl-2 level （P<0.01）， and down-regulated the mRNA levels of Fas， FasL， and Fadd and the protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.05， P<0.01） in the hippocampus. Low-dose Hei Xiaoyaosan decreased the apoptosis rate in the cortex and hippocampus （P<0.05， P<0.01）， lowered the Bcl-2 level （P<0.01）， and down-regulated the mRNA levels of FasL and Fadd （P<0.05） and the protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.05） in the hippocampus. ConclusionHei Xiaoyaosan can protect neurons in the cortex and hippocampus of AD rats by inhibiting the apoptosis mediated by the Fas/FasL/Caspase-8/Caspase-3 signaling pathway.

Salvianolic Acid F Regulates Bax/Caspase-3/GSDME Signaling Pathway to Inhibit Pyroptosis of HK-2 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of salvianolic acid F （Sal F） in repairing the high glucose-induced injury in human kidney-2 （HK-2） cells via the B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）/cysteinyl aspartate-specific proteinase 3 （Caspase-3）/gasdermin-E （GSDME） pathway. MethodThe cell counting kit-8 （CCK-8） was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations （2.5， 5， 10， 20 μmol·L-1） of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods. The levels of lactate dehydrogenase （LDH） and interleukin-1β （IL-1β） in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay （ELISA） kit， respectively. Flow cytometry combined with Annexin V-FITC/propidium iodide （PI） and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose. Western blotting was employed to determine the protein levels of Bax， Bcl-2， cytochrome C， cysteinyl aspartate-specific proteinase （Caspase）-9， Caspase-3， and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F. The 2，7-dichlorodihydrofluorescein diacetate fluorescence probe （DCFH-DA） and mitochondrial membrane potential assay kit （JC-1） were used to determine the production of reactive oxygen species （ROS） and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F. ResultCompared with the blank group， the model group showed decreased cell viability （P<0.01）， elevated levels LDH and IL-1β， increased proportion of PI-positive cells （P<0.01）， up-regulated protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME （P<0.01）， down-regulated protein level of Bcl-2 （P<0.01）， decreased mitochondrial membrane potential， and excessive ROS accumulation. Compared with the model group， Sal F repaired the high glucose-induced injury in HK-2 cells （P<0.05）， lowered the levels of LDH and IL-1β （P<0.05， P<0.01）， and decreased the proportion of PI-positive cells （P<0.01）. In addition， Sal F down-regulated the protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME and up-regulated the protein level of Bcl-2 （P<0.05， P<0.01）， increased the mitochondrial membrane potential， and decreased the accumulation of ROS in HK-2 cells. ConclusionSal F can reduce the production of ROS， restore the balance of mitochondrial membrane potential， and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury in HK-2 cells.