Role of NLRP3/Caspase-3 in myocardial ischemia/reperfusion injury in rats / 中华老年心脑血管病杂志

Objective To explore the role of NLRP3/Caspase-3 in myocardial apoptosis induced by ischemia/reperfusion(IR)injury and its effect on myocardiocyte autophagy in rats.Methods A total of 60 SPF-grade male rats were randomly divided into sham operation,model,and nimodip-ine treatment groups,with 20 rats in each group.Rat model of myocardial IR injury was estab-lished in the rats of the two latter groups.Cardiac function was assessed,and the levels of myocar-dial enzymes and cytokines were measured.Additionally,myocardial pathological changes were de-tected using HE staining.Furthermore,flow cytometry was utilized to determine the apoptotic rate of myocardiocytes,and the autophagosomes were counted under transmission electron micro-scope.Moreover,the expression of NLRP3 and Caspase-3 was measured using RT-PCR and West-ern blotting.Results Significant differences were observed in left ventricular end diastolic pres-sure,left ventricular systolic pressure,maximal rate of rise and fall in left ventricular pressure,ap-optotic rate of myocardial cells,and levels of TNF-α,IL-6,CK,AST and LDH in the three groups(P＜0.01).Notably,both the model group and nimodipine treatment group exhibited significantly higher autophagosome than the sham operation group(10.55±1.87 and 6.32±1.43 vs 3.45±0.67 units,P＜0.01),and the nimodipine group displayed a significantly lower autophagosome count than the model group(P＜0.01).The mRNA and protein levels of NLRP3 and Caspase-3 were notably higher in the model group and nimodipine group than the sham operation group(P＜0.01),and in the model group than the nimodipine group(P＜0.01).Conclusion Myocardial IR injury in rats can increase myocardiocyte apoptosis,reduce cardiac function,induce inflammatory response,and enhance autophagosome formation,which is related to the abnormal high expression of NLRP3/Caspase-3.

Expression of pyroptosis related molecules in primary gouty arthritis and its clinical value / 中华风湿病学杂志

Objective:To explore the molecular mechanism of cell death of the peripheral blood mononuclear cells (PBMCs) of patients with primary gouty arthritis, and provide new idea for the treatment of gout.Methods:Peripheral blood samples and clinical data were collected from 30 patients with acute gout (AG), 30 patients with intermittent gout (IG) and 40 healthy controls(HC). Real-time fluorescence quantitative detection of cell apoptosis related molecules, including the mRNA expression level of nucleotide binding oligomerization domain like domain like receptor protein 3(NLRP3), cysteine aspartic proteinase-1/4/5 (caspase-1/4/5), Gasdermin A/B/C/E. And NLRP3, precursor caspase-1 (pro-caspase-1), clipped caspase-1 (caspase-1 + p10), Gasdermin D(GSDMD), N segment GSDMD (GSDMD-N), precursor IL-1β (pro IL-1β), mature IL-1β (clevated IL-1β)were detected by western blot. The measurement data of normal or approximate normal distribution were analyzed by independent sample t test or one-way variance analysis (ANOVA), the measurement data of non-normal distribution were analyzed by Mann-Whitney U test or Kruskal-Wallis H test, and the counting data was compared by Chi-square test. Pearson's correlation analysis was used for the continuous variables with normal distribution, and Spearman's correlation analysis was used for the continuous variables with non-normal distribution. The logistic regression analysis was used to assess risk factors. Results:① There were no significant differences in MPR and BMI between AG and IG ( χ2=0.64, P=0.426; t=0.04, P=0.972), and there was significant difference in disease course [25.0 (9.8, 63.0), 54.0 (33.0, 102.0)mouth, Z=2.01, P=0.044]. Comparison of labora-tory parameters: there were statistical significant differences in ESR between AG and IG ( t=5.24, P<0.001), eGFR, GR, LY, RBC, HCT, UA, Creatinin, ALT and AST. ② In the three groups, the expression lev-els of caspase-1, GSDMC, GSDMD, GSDME, NLRP3 mRNA were statistically significantly different. In AG and IG groups, mRNA expression levels of caspase-1 (1.55±0.62), (1.58±0.62), GSDMD (4.7±1.4), (3.5±1.53), NLRP3 [2.63(2.03, 4.10), 2.39(1.57, 3.49)] were higher than those of the HC group [(1.24±0.59), 1.16±0.71, 1.16 (0.50, 2.34)] ( P=0.037, P=0.023, P<0.001, P<0.001, P<0.001, P<0.001). In IG group, mRNA expression levels of GS-DMD (3.53±1.53) were lower than those of AG group (4.68±1.43) ( P<0.001).The mRNA expression levels of GS-DMC and GSDME [0.57(0.33, 0.78), (0.32±0.15)]were lower than those of the HC group [0.80 (0.47, 1.86), (1.06 ± 0.36) ( P=0.004, P<0.001), and the mRNA expression levels of GSDME (0.62±0.29) in the IG group were lower ( P=0.004, P<0.001), However, in the IG group, GSDMC and GSDME [0.87 (0.51, 1.53), (0.62±0.29)] were higher than those in the AG group [0.57 (0.33, 0.78), (0.32±0.15)] ( P=0.003, P<0.001). ③ The expression levels of NLRP3, pro-caspase-1, caspase-1 + p10, GSDMD, GSDMD-N, pro-IL-1β, clevated IL-1β protein were statistically different among the three groups [( F=50.04, P<0.001; F=9.65, P=0.013; F=30.71, P=0.001; F=7.38, P=0.024; F=23.66, P=0.001; F=30.11, P=0.001; F=6.01, P=0.036]. The expression of NLRP3 protein in the AG group (1.14±0.12) was significantly higher than that in the IG and HC group (0.35±0.18), (0.17±0.03) (all P=0.001), the expression levels of Pro caspase-1, caspase-1+p10 protein in the AG (1.11±0.15), (0.93±0.38) and IG (0.98±0.14), (1.14±0.17) group were higher than those in the HC (0.42±0.28), (0.29±0.16) ( P=0.006, P=0.015). The expression levels of GSDMD protein in the AG group (1.04±0.16) were higher than those in the IG and HC group(0.53±0.26), (0.39±0.22) ( P=0.029, P=0.011). The expression level of GSDMD-N protein in the AG and IG group (0.97±0.06), (0.90±0.04) was higher than that in the HC (0.27±0.23) ( P=0.001, P=0.001). The expression level of pro-IL-1β protein in the AG group (1.01±0.06) was significantly higher than that in the IG and HC group (0.32±0.14), (0.64±0.11) ( P<0.001, P=0.006), but lower than that in the HC (0.64±0.11) ( P=0.011). The expression of clevated IL-1β protein was higher in the AG group (1.08±0.20) than in the HC group (0.33±0.24) ( P=0.014). ④ Negative correlation between NLRP3, GSDMD and LY ( r=-0.32, P=0.001; r=-0.24, P=0.017) and positive correlation between GSDMD and WBC, GR ( r=0.43, P<0.001; r=0.23, P=0.019) were found and Logistic regression analysis showed that the GSDMD and NLRP3 were risk factors for AG [ OR ( 95%CI)=11.29 (3.92, 32.48), P<0.001; OR( 95%CI)=2.21(1.00, 4.85), P=0.049]. GSDMD was risk factor for IG [ OR( 95%CI)=6.84(2.52, 18.53), P<0.001]; While GSDMD was the protective factor for IG [ OR( 95%CI)=0.61(0.41, 0.30), P=0.013]. Conclusion:The expression's of NLRP3, Caspase-1 and GSDMD are increased in PBMCs of AG patients, while the expression's of GSDMC and GSDME are decreased. NLRP3/Caspase-1/GSDMD may be associated with the onset of acute gouty arthritis.

The underlying mechanism of autophagy regulating liver injury with obstructive jaundice in Sprague-Dawley rats / 中华肝胆外科杂志

Objective:To investigate the effect of autophagy on liver injury with obstructive jaundice in Sprague-Dawley (SD) rats and its underlying mechanism.Methods:Thirty-five healthy male SD rats, SPF grade, aged 6-8 weeks, weighting 200-300 g, were divided into 5 groups with 7 rats in each group, including sham group (simple free common bile duct, without ligation, intraperitoneal injection of normal saline), obstructive jaundice (OJ) group (established by common bile duct ligation, intraperitoneal injection of normal saline), OJ group with 3-MA, OJ group with Rapamycin, and OJ group with 3-MA and VX-765. Morphological changes in liver tissues were analyzed with HE staining. Expression of autophagy-related protein Atg5 was detected by immunohistochemistry staining. Liver function was analyzed by automatic biochemical instrument and the level of serum interleukin (IL)-18 was detected using ELISA assay. Protein levels of autophagy related-proteins and endoplasmic reticulum stressed (ERs)-related apoptosis proteins were detected by Western Blot.Results:The relative expression of autophagy related protein Atg5 in OJ group was significantly higher than that in sham group [(5.0±1.0) vs. (2.8±1.3), t=-3.00, P<0.05]. Compared with sham group, the activity of autophagy was enhanced and the protein levels of Caspase-1/p-65 and IL-18 were significantly increased in OJ group. At the same time, apoptosis was induced by activating ERs. In OJ group, the autophagy inducer 3-MA improved the expression levels of Caspase-1/p-65 and IL-18, and aggravate liver injury. While after applying the autophagy agonist Rapamycin in OJ rat models, the expression of Caspase-1/p-65 and IL-18 was repressed and liver damage was also reduced. In addition, in rat OJ groups with 3-MA, inhibition of Caspase-1 by VX-765 could down regulate the expression of Caspase-1/p-65 and IL-18, and protect against liver injury. Conclusions:Both ERs related apoptosis and autophagy were activated after ligation of common bile duct. Besides, activation of autophagy could reduce OJ-induced liver injury in SD rats by inhibiting the Caspase-1/p-65 inflammatory pathway.

Progress of pyroptosis in gynecological diseases / 肿瘤研究与临床

Pyroptosis is a fairly new type of cell death that is frequently mediated by inflammasomes and is strongly related to Gasdermin protein family and cysteinyl aspartate specific proteinase (caspase). The importance of pyroptosis-related molecules in the occurrence and progression of gynecological diseases cannot be overstated. This article reviews the related mechanism of cell pyroptosis, and the relationship between pyroptosis and benign and malignant gynecological diseases, so as to provide references for the research of the diagnosis and treatment of gynecological diseases, as well as the development of targeted drugs.

Apoptosis (programmed cell death) and its signals - A review / Apoptose, morte celular programada e seus sinais: uma revisão

Abstract Apoptosis is a sequential order of cell death occurring regularly to ensure a homeostatic balance between the rate of cell formation and cell death. However, a misplaced of this balancing function can contribute to an abnormal cell growth / proliferation or autoimmune disorders etc. Apoptosis is therefore said to be crucial from the point of development of an embryo throughout the growth of an organism contributing to the renewal of tissues and also the getting rid of inflammatory cells. This review seeks to elaborate on the recent overview of the mechanism involved in apoptosis, some element and signal contributing to its function and inhibition together with how their malfunction contribute to a number of cancer related cases.

Resumo A apoptose é uma ordem sequencial de morte celular que ocorre regularmente para garantir um equilíbrio homeostático entre a taxa de formação e a morte celular. No entanto, um desequilíbrio dessa função pode contribuir para um crescimento/proliferação celular anormal ou distúrbios autoimunes. A apoptose é, portanto, considerada crucial do ponto de desenvolvimento de um embrião ao longo do crescimento de um organismo que contribui para a renovação dos tecidos e também a eliminação de células inflamatórias. Esta revisão procura elaborar a recente visão geral do mecanismo envolvido na apoptose, alguns elementos e sinais que contribuem para sua função e inibição, além de como o mau funcionamento deles contribui para vários casos relacionados ao câncer.

The role of inflammatory molecules related to pyroptosis pathway in the pathogenesis of atopic dermatitis / 国际儿科学杂志

Atopic dermatitis(AD)is a common skin disease in pediatrics.It is a chronic inflammatory skin disease related to allergic diathesis that interacts with genetic and environmental factors.In about 60% of patients, the onset is before the age of one.With the deepening of research on the pathogenesis of AD, current research shows that pyroptosis-related inflammatory molecules may be involved in the occurrence and development of AD.Pyroptosis is a kind of regulated cell death accompanied by the release of inflammatory cytokines.It has been proven to play an important role in diseases such as atherosclerosis, diabetes, asthma, and inflammatory bowel disease.This article summarizes the role of inflammatory molecules related to pyroptosis pathway in the pathogenesis of atopic dermatitis, in order to deepen the understanding of the pathogenesis of atopic dermatitis and explore new therapies for it.

Peanut testa extracts enhance anticancer effect of cisplatin against human cholangiocarcinoma cells via modulation of histone deacetylase inhibitory activity

Objective: To investigate the effect of combination treatments of cisplatin and KK4 and ICG15042 peanut testa extracts against cholangiocarcinoma cells in vitro.Methods: The growth inhibition, cell cycle arrest and apoptosis of cholangiocarcinoma cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry analysis, respectively. The levels of proteins involved in apoptosis were assessed using Western blotting assays. The caspase activity was assessed using a colorimetric caspase activity assay. Results: Cisplatin and peanut (KK4 and ICG15042) testa extracts inhibited the growth of cholangiocarcinoma cell lines (KKU-M214 and KKU-100 cells) in a dose- and time-dependent manner. The combination treatments reduced cell viability and induced apoptosis of cholangiocarcinoma cells more efficiently than single-drug treatments. Cancer cell death synergistically mediated by cisplatin and peanut testa extracts was observed in KKU-M214 cells (combination index < 1.0) but not in KKU-100 cells (combination index > 1.0). The combination treatments also increased the sub-G1 population and caused KKU-M214 cell cycle arrest at S and G2/M phases, which were the combined effects of cisplatin (S phase arrest) and peanut testa extracts (G2/M phase arrest). In addition, pERK1/2, Ac-H3, Bcl-2 and proteins related to apoptosis, including Bax and caspases 3, 8, 9, exhibited enhanced expression in KKU-M214 cells. The combination treatments caused down-regulation of p53, whereas the expression of p21 was fairly constant when compared with cisplatin single drug treatment. Conclusions: Peanut testa extracts in combination with cisplatin synergistically reduce cell viability and induce apoptosis through stimulation of caspases 3, 8 and 9 in KKU-M214 cells.

Effect of electroacupuncture on the learning and memory abilities in type 2 diabetic model rats with cognitive impairment / 针灸推拿医学（英文版）

Objective: To observe the effects of electroacupuncture (EA) on the protein and gene expressions of Bax, Caspase-3 and Bcl-2 in cerebral cortex of type 2 diabetic rats with cognitive impairment (CI), and to explore the mechanism of EA in improving the learning and memory abilities. Methods: A total of 100 Sprague-Dawley (SD) rats were divided into a normal group (n=10) and a model group (n=90) by random number table method. Rats in the model group were intraperitoneally injected with a small dose of streptozotocin (STZ) to establish the type 2 diabetic models, after being fed with high-fat and high-sugar diet for 1 month. Twenty CI rats were selected from the 50 successful model rats by the Morris water maze (MWM) test and randomly divided into a model group and an EA group according to the blood glucose level and MWM data (n=10). Rats in the EA group received acupuncture at Zusanli (ST 36), Neiting (ST 44) and Yishu (Extra), of which Zusanli (ST 36) and Neiting (ST 44) were stimulated by EA apparatus, 20 min/time, once a day for 6 d a week and 4 consecutive weeks. The rats in the model and the normal groups were fixed without treatment. After 4-week treatment, the random blood glucose level of the rats was measured; the learning and memory abilities of rats were measured by MWM; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptotic cells; Western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the protein and gene expressions of Bax, Caspase-3 and Bcl-2 in cerebral cortex. Results: After modeling, the random blood glucose level and the escape latency tested by MWM were significantly increased, and the number of crossing the platform tested by the MWM was decreased in the EA and model groups, and were significantly different from those in the normal group (P<0.05 or P<0.01), while the differences between the model group and the EA group were not statistically significant (all P>0.05). After 4-week treatment, the random glucose level and the escape latency tested by MWM were significantly increased (both P<0.05), and the number of crossing the original platform tested by the MWM was significantly reduced (P<0.01), the protein and gene expressions of Bax and Caspase-3 were significantly increased (all P<0.001), the protein and gene expressions of Bcl-2 were significantly reduced (both P<0.001), and the number of neuron apoptosis was significantly increased (P<0.001) in the model group than in the normal group; the random blood glucose level was significantly reduced (P<0.05), the escape latency tested by MWM was significantly shortened (P<0.05), and the number of crossing the original platform tested by MWM was significantly increased (P<0.05), the protein and gene expressions of Bax and Caspase-3 were significantly reduced (all P<0.001), the protein and gene expressions of Bcl-2 were significantly increased (both P<0.001), and the number of neuron apoptosis was significantly reduced (P<0.001) in the EA group than in the model group. Conclusion: EA can improve the learning and memory damages induced by type 2 diabetic model rats with CI; the action mechanism may be achieved via anti-apoptosis.

Peanut testa extracts enhance anticancer effect of cisplatin against human cholangiocarcinoma cells via modulation of histone deacetylase inhibitory activity

Objective: To investigate the effect of combination treatments of cisplatin and KK4 and ICG15042 peanut testa extracts against cholangiocarcinoma cells in vitro. Methods: The growth inhibition, cell cycle arrest and apoptosis of cholangiocarcinoma cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry analysis, respectively. The levels of proteins involved in apoptosis were assessed using Western blotting assays. The caspase activity was assessed using a colorimetric caspase activity assay. Results: Cisplatin and peanut (KK4 and ICG15042) testa extracts inhibited the growth of cholangiocarcinoma cell lines (KKU-M214 and KKU-100 cells) in a dose- A nd time-dependent manner. The combination treatments reduced cell viability and induced apoptosis of cholangiocarcinoma cells more efficiently than singledrug treatments. Cancer cell death synergistically mediated by cisplatin and peanut testa extracts was observed in KKU-M214 cells (combination index 1.0). The combination treatments also increased the sub-G1 population and caused KKU-M214 cell cycle arrest at S and G2/M phases, which were the combined effects of cisplatin (S phase arrest) and peanut testa extracts (G2/M phase arrest). In addition, pERK1/2, Ac-H3, Bcl-2 and proteins related to apoptosis, including Bax and caspases 3, 8, 9, exhibited enhanced expression in KKU-M214 cells. The combination treatments caused down-regulation of p53, whereas the expression of p21 was fairly constant when compared with cisplatin single drug treatment. Conclusions: Peanut testa extracts in combination with cisplatin synergistically reduce cell viability and induce apoptosis through stimulation of caspases 3, 8 and 9 in KKU-M214 cells.

Chronic alcoholism associated with diabetes induced apoptosis in the corpus cavernosum of rats

Abstract Purpose To evaluate the effects of alcohol exposure and diabetes on apoptotic process in the corpus cavernosum. Methods Forty eight male Wistar rats were divided into four groups: control, diabetic, alcoholic and diabetic-alcoholic. Samples of the corpus cavernosum were prepared to study protein expression of apoptotic genes (Caspases-3 and 9) by immunohistochemistry and Real-Time PCR. Results The immunoreactivity of Caspases-3 and -9 was diffuse and higher in the treated groups though there was no significant difference between the experimental groups, only when compared with the control group. An increase was observed in the gene expression of Caspases-9 in the diabetic and ethanol-diabetic groups when compared with control and ethanol groups. Conclusions The association of these factors (ethanol and diabetes) probably can affect the apoptosis mechanism in lesions of the cavernous tissue in the rat penis. Both gene and protein expression of Caspase-9 in diabetic and ethanol-diabetic groups suggest the involvement of the apoptosis cascade from this study model.

Alpha-hederin induces the apoptosis of oral cancer SCC-25 cells by regulating PI3K/Akt/mTOR signaling pathway

BACKGROUND: Oral cancer is one of the common malignant tumors of the head and neck. However, current treatments have numerous side effects, and drugs from natural sources may have better therapeutic potential. This research investigated the induction of apoptosis by α-hederin (α-HN), a constituent of Pulsatilla chinensis (Bunge) Regel, in the oral cancer cell line SCC-25 and its underlying mechanism. RESULTS: SCC-25 cells were treated with 50, 100, and 200 µmol/L α-HN. Cell proliferation; extent of apoptosis; activities of caspases-3, 8, and 9; and the expression of Bcl-2, Bax, phosphorylated (p)-phosphoinositide 3-kinase (PI3K), p-Akt, and p-mammalian target of rapamycin (mTOR) proteins were determined using the 3-(4,5)-2-thiazole-(2,5)-diphenyl tetrazolium bromide, flow cytometry, caspase activity detection kits, and western blot assays, respectively. The results showed that the proliferation of SCC-25 cells in the α-HN-treated groups decreased significantly, and the inhibitory effect was time and concentration dependent. Compared with cells in the control group, the extent of apoptosis increased significantly, caspase-3 and -9 activities were significantly enhanced, and the Bcl-2 level was lowered and the Bax level was elevated significantly in SCC-25 cells treated with α-HN for 48 h (P b 0.05). The expression of p-PI3K, p-Akt, and p-mTOR was also significantly lower in SCC-25 cells treated with α-HN than that in the control group (P b 0.05). CONCLUSION: These results indicate that α-HN can inhibit proliferation and induce apoptosis of SCC-25 cells and may exert these effects by inhibiting the PI3K/Akt/mTOR signaling pathway.

Plasma Levels of K18 Fragments Do Not Correlate with Alcoholic Liver Fibrosis

BACKGROUND/AIMS: Noninvasive markers of liver fibrosis in alcoholic liver disease (ALD) are crucial to establish early intervention. Previous studies have suggested that plasma levels of cleaved keratin-18 (K18; M30) fragments can predict the severity of liver disease. The aim of this study was to correlate plasma M30 levels with stages of liver fibrosis in ALD. METHODS: Patients with ALD (n=139, 79.1% males) and liver histology were included, and plasma samples were collected to quantify plasma M30 levels. Patients were stratified into five groups by fibrosis stage (F0=14; F1=15; F2=35; F3=17; and F4=58) according to the Kleiner score. Differences between groups were evaluated using the chi-square test or analysis of variance. Trends by fibrosis stage were calculated by logistic regression analysis, and sensitivity, specificity and positive and negative predictive values were determined. RESULTS: There were no significant differences in M30 levels among fibrosis stages. The correlation between plasma M30 levels and fibrosis was poor (Pearson’s correlation coefficient=0.13, Spearman rho=0.20 [p=0.02]), and M30 levels did not correlate with alcohol-specific histological features. However, significant correlations of M30 levels with aspartate aminotransferase (Spearman rho=0.653, p 200 U/L reveal a sensitivity for predicting cirrhosis of 84.5% with a negative predictive value of 73.5%. CONCLUSIONS: Plasma M30 levels are often elevated in ALD and correlate with serum transaminases but do not reflect fibrosis. The usefulness as a prognostic marker awaits evaluation in prospective studies.

Relationship between TIPE2 and caspase-11 during pyroptosis in macrophages of mice / 中华麻醉学杂志

Objective@#To evaluate the relationship between tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) and caspase-11 during pyroptosis in macrophages of mice.@*Methods@#J774A.1 macrophages of mice were divided into 4 groups (n=6 each) using a random number table method: non-specific siRNA (Scr-siRNA) group (S group), Scr-siRNA plus LPS/ATP group (S+ LPS/ATP group), TIPE2-siRNA group (T group) and TIPE2-siRNA plus LPS/ATP group (T+ LPS/ATP group). The corresponding siRNA and Lipofectamine2000 transfection reagents were added to each group, and transfection was performed for 24-48 h, and in addition LPS 1.0 μg/ml was then added, cells were incubated for 5 h, then ATP 5.0 mmol/L was added, and cells were incubated for 1 h in S+ LPS/ATP and T+ LPS/ATP groups.Cells were collected to detect the expression of TIPE2, caspase-11, NOD-like receptor familypyrin domain containing 3 (NLRP3) and caspase-1 (by Western blot). The supernatant was collected for determination of lactic dehydrogenase (LDH) and myeloperoxidase (MPO) activities and concentrations of interleukin-1beta (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay).@*Results@#Compared with group S, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group S+ LPS/ATP (P<0.05). Compared with group T, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+ LPS/ATP (P<0.05). Compared with group S+ LPS/ATP, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+ LPS/ATP (P<0.05).@*Conclusion@#TIPE2 inhibits pyroptosis in macrophages through down-regulating the expression of caspase-11 in mice.

Relationship between TIPE2 and caspase-11 during pyroptosis in macrophages of mice / 中华麻醉学杂志

Objective To evaluate the relationship between tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) and caspase-11 during pyroptosis in macrophages of mice.Methods J774A.1 macrophages of mice were divided into 4 groups (n =6 each) using a random number table method:nonspecific siRNA (Scr-siRNA) group (S group),Scr-siRNA plus LPS/ATP group (S+LPS/ATP group),TIPE2-siRNA group (T group) and TIPE2-siRNA plus LPS/ATP group (T+LPS/ATP group).The corresponding siRNA and Lipofectamine2000 transfection reagents were added to each group,and transfection was performed for 24-48 h,and in addition LPS 1.0 μg/ml was then added,cells were incubated for 5 h,then ATP 5.0 mmol/L was added,and cells were incubated for 1 h in S + LPS/ATP and T + LPS/ATP groups.Cells were collected to detect the expression of TIPE2,caspase-11,NOD-like receptor familypyrin domain containing 3 (NLRP3) and caspase-1 (by Western blot).The supernatant was collected for determination of lactic dehydrogenase (LDH) and myeloperoxidase (MPO) activities and concentrations of interleukin-1beta (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay).Results Compared with group S,the expression of TIPE2 was significantly down-regulated,the expression of caspase-11,NLRP3 and caspase-1 was up-regulated,and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group S+LPS/ATP (P＜0.05).Compared with group T,the expression of TIPE2 was significantly down-regulated,the expression of caspase-11,NLRP3 and caspase-1 was up-regulated,and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+LPS/ATP (P＜0.05).Compared with group S+LPS/ATP,the expression of TIPE2 was significantly down-regulated,the expression of caspase-11,NLRP3 and caspase-1 was up-regulated,and and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+LPS/ATP (P＜0.05).Conclusion TIPE2 inhibits pyroptosis in macrophages through down-regulating the expression of caspase-11 in mice.

Efficacy of phosphocreatine pre-administration on XIAP and Smac in ischemic penumbra of rats with focal cerebral ischemia reperfusion injury

Abstract Purpose: To observe the efficacy of phosphocreatine pre-administration (PCr-PA) on X-linked inhibitor of apoptosis protein (XIAP), the second mitochondia-derived activator of caspase (Smac) and apoptosis in the ischemic penumbra of rats with focal cerebral ischemia-reperfusion injury (CIRI). Methods: A total of 60 healthy male Sprague Dawley (SD) rats were randomly divided into three groups (n=20): group A (the sham operation group), group B <intraperitoneally injected with 20 mg/kg (10 mg/ml) of saline before preparing the ischemia-reperfusion (IR) model>, and group C <intraperitoneally injected with 20 mg/kg (10 mg/ml) of PCr immediately before preparing the IR model>. After 24 h for reperfusion, the neurological function was evaluated and the tissue was sampled to detect expression of XIAP, Smac and caspase-3 positive cells in the ischemic penumbra so as to observe the apoptosis. Results: Compared with group B, neurological deficit scores, numbers of apoptotic cells, expression of Smac,caspase-9 and the numbers of Caspase-3 positive cells were decreased while expression of XIAP were increased in the ischemic penumbra of group C. Conclusions: Phosphocreatine pre-administration may elicit neuroprotective effects in the brain by increasing expression of X-linked inhibitor of apoptosis protein, reducing expression of second mitochondia-derived activator of caspase, and inhibiting the apoptosis in the ischemic penumbra.

Effects of curcumin on expression of caspase-3 , caspase-8 and caspase-9 in PC12 cells induced by Aβ25-35 / 中国病理生理杂志

AIM:To investigate the effects of curcumin on the viability ,the lactate dehydrogenase(LDH)re-lease,the apoptosis,and the activity and the expression levels of caspase-3,caspase-8 and caspase-9 of rat adrenal pheo-chromocytoma PC12 cells induced by β-amyloid protein 25-35(Aβ25-35 ).METHODS:The PC12 cells were treated with Aβ25-35.The viability and LDH release rates were measured by MTT assay and LDH kit ,respectively.The cells were ran-domly divided into blank control group ,model group,curcumin 10μmol/L group and curcumin 20μmol/L group.The ap-optotic rates were evaluated by flow cytometry with Annexin V-FITC/PI staining.The activities and expression levels of caspase-3,caspase-8 and caspase-9 were detected by colorimetric method and Western blot analysis.RESULTS:Com-pared with model group ,curcumin significantly increased the viability ,and decreased the LDH release rates and the apop-totic rates of the PC12 cells treated with Aβ25-35(P<0.01).Compared with model group,curcumin significantly decreased the activity and expression levels of caspase-3,caspase-8 and caspase-9(P<0.05 or P<0.01).CONCLUSION:Cur-cumin inhibits Aβ25-35-induced apoptosis of PC12 cells by inhibiting the expression of caspase-3,caspase-8 and caspase-9.

Effects of turmeric volatile oil combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431 and their mechanisms / 中华皮肤科杂志

Objective To evaluate the effects of turmeric volatile oil (TVO) combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431,and to explore their mechanisms.Methods Some cultured A431 cells at exponential growth phase were divided into several groups to be treated with 5,10,20,40 and 80 mg/L TVO,as well as high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 1％ dimethyl sulfoxide (DMSO,control group),respectively.After 24-hour treatment,cell counting kit 8 (CCK8) assay was performed to estimate the proliferative activity of A431 cells in the above groups.Some other A431 cells were divided into 4 groups:control group treated with high-glucose DMEM containing 1％ DMSO,TVO group treated with 40 mg/LTVO,cisplatin group treated with 10 mg/L cisplatin,and TVO + cisplatin group treated with 40 mg/L TVO and 10 mg/L cisplatin.After 24-hour treatment,CCK8 assay was performed to estimate the cellular proliferative activity,inverted microscopy to observe changes in cell morphology,fluorescence microscopy to detect cell apoptosis after acridine orange (AO)/ethidium bromide (EB) double-staining,colorimetry to evaluate the activity of Caspase-3 and Caspase-9,and Western blot analysis to determine the protein expression of Caspase-3 and p-glycoprotein.Results After 24-hour treatment with 5,10,20,40 and 80 mg/L TVO,the cell proliferation rates were inhibited by (12.83 ± 6.4)％,(16.27 ± 11.4)％,(21.61 ± 9.1)％,(33.11 ± 2.0)％ and (46.00 ± 3.3)％ respectively,and the inhibition rates were all significantly higher in these groups than in the control group (4.03％ ± 1.4％,all P ＜ 0.05).The 50％ inhibitory concentration (IC50) of TVO at 24 hours was (61.66 ± 1.03) mg/L.Compared with the control group,the proliferation inhibition rates significantly increased in the TVO group,cisplatin group and TVO + cisplatin group (all P ＜ 0.05),suggesting that the combination of TVO and cisplatin showed synergistic inhibitory effects with a combination index of 1.366.Moreover,A431 cells turned round to different extents and became apoptotic in the TVO group and cisplatin group,and the TVO + cisplatin group showed obviously decreased number of cells and a large number of cell debris.The TVO + cisplatin group also showed significantly increased activity of Caspase-3 (1.520 ± 0.115) and Caspase-9 (2.760 ± 0.297) as well as protein expression of Caspase-3 (1.482 ± 0.016) compared with the TVO group (Caspase-3 activity:1.117 ± 0.095;Caspase-9 activity:1.259 ± 0.059;Caspase-3 protein expression:1.156 ± 0.006,all P ＜ 0.01) and cisplatin group (Caspase-3 activity:1.381 ± 0.089;Caspase-9 activity:1.829 ± 0.171;Caspase-3 protein expression:1.296 ± 0.021,all P ＜ 0.01),but significantly decreased p-glycoprotein expression (0.528 ± 0.014) compared with the TVO group (1.311 ± 0.011,P ＜ 0.01) and cisplatin group (1.169 ± 0.012,P ＜ 0.01).Conclusion TVO combined with cisplatin can synergistically inhibit the proliferation of A431 cells and induce cell apoptosis,which may be associated with activation of the caspase system and decreased expression of pglycoprotein.

Icariin reduces human colon carcinoma cell growth and metastasis by enhancing p53 activities

Icariin has been reported to possess high anticancer activity. Colon carcinoma is one of the leading causes of cancer-related mortality worldwide. Here, the anticancer activity of icariin against HCT116 colon carcinoma cells and the possible underlying mechanism were studied. The trypan blue staining assay, wound healing assay, clonogenic assay, CCK-8 assay, and Annexin V-FITC/PI double staining method were carried out to determine the changes of HCT116 cell growth and migration. mRNA and protein expressions were determined by quantitative real-time PCR and western blot, respectively. Moreover, small interfering RNA (siRNA) plasmid was used to examine the role of p53 in icariin-induced apoptosis in HCT116 cells. Icariin significantly suppressed colon carcinoma HCT116 cells by decreasing migration and viability, and simultaneously promoting apoptosis. Icariin exerted the anti-tumor effect in a dose-dependent manner by up-regulating p53. During treatment of icariin, p-p53, p21, and Bax levels increased, and Bcl-2 level decreased. Short time treatment with icariin induced DNA damage in HCT116 cells. Furthermore, the cytotoxicity of icariin was decreased after p53 knockdown or by using caspase inhibitors. p53 was involved in activities of caspase-9 and caspase-3. Icariin repressed colon carcinoma cell line HCT116 by enhancing p53 expression and activating p53 functions possibly through Bcl-2/Bax imbalance and caspase-9 and -3 regulation. Icariin treatment also induced DNA damage in HCT116 cells.

Apoptosis mechanism of gastric cancer cellSGC-7901 induced by Omphalia lapidescens protein pPeOp / 中国药理学通报

Aim To investigate the apoptosis mechanism of human gastric cancer cell SGC-7901 induced by Omphalia lapidescens protein pPeOp.Methods CCK-8 and flow cytometry were used to detect the inhibitory effect of different concentrations of pPeOp(30, 60, 90 mg·L-1) on SGC-7901.The mRNA and protein expression of TNF-R1, Fas/FasL, Bcl-2, caspase-3 and caspase-8 were detected by qRT-PCR and Western blot.Results SGC-7901 cells were treated with different concentrations of pPeOp(30, 60, 90 mg·L-1) for 24 h.CCK-8 test showed that there was no significant difference between PVP group and the control group.The survival rate of the 5-Fu group was(53.71±7.34)% (P<0.05).The survival rates of pPeOp group(30, 60, 90 mg·L-1) were(80.95±6.25)%, (53.48±5.70)% and(44.61±6.50)%(r=0.984,P=0.016),respectively.Flow cytometry showed that the apoptosis rate of PVP group had no significant difference with control group, and the apoptosis rate of 5-Fu group was about(39.30±3.34)%(P<0.05).The apoptotic rates of pPeOp group(30, 60, 90 mg·L-1) were(10.90±1.25)%, (28.80±2.70)% and (32.00±3.50)%,respectively(P<0.05).The mRNA and protein expression levels of Bcl-2 were down-regulated,whereas the expression of TNF-R1, Fas/FasL, caspase-3 and caspase-8 were significantly up-regulated(P<0.05).Conclusions pPeOp can significantly inhibit the proliferation of gastric cancer cell line SGC-7901 and induce apoptosis in a dose-dependent manner.Death receptor pathway and mitochondrial pathway may be related to pPeOp-induced apoptosis of gastric cancer SGC-7901.

Effects of acupuncture plus mild hypothermia on apoptosis-related factors in rats with cerebral ischemia-reperfusion / 针灸推拿医学（英文版）

Objective: To investigate the effect of acupuncture plus mild hypothermia on neurological function impairment score, cerebral infarct size and apoptosis-related factors in cerebral ischemia reperfusion injury (CIRI) rats.Methods: Sixty healthy male Sprague-Dawley (SD) rats were routinely reared for 1 week. Ten rats were randomly selected as the sham operation group and 10 rats as the blank control group, while the remaining 40 rats were subjected to preparing the middle cerebral artery occlusion (MCAO) model by modified filament occlusion method. The 40 MCAO rats were further randomly divided into a model group, an acupuncture group, a mild hypothermia group and an acupuncture plus mild hypothermia group, with 10 rats in each group. Rats in the sham operation group, the blank control group and the model group did not accept treatment except binding; rats in the acupuncture group received acupuncture treatment; rats in the mild hypothermia group received mild hypothermia treatment; rats in the acupuncture plus mild hypothermia group received acupuncture and mild hypothermia treatment. 72 h after the treatment, neurological function impairment score was performed; the infarct area ratio was determined by 2,3,5-tripheyl tetrazolium chloride (TTC) staining; apoptosis of brain cells was observed by TUNEL method; the expressions of Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry.Results: Compared with the blank control group and the sham operation group, the neurological function impairment score, cerebral infarct area ratio, apoptosis, and the expressions of Bax and Caspase-3 in the model group were significantly increased, while the expression of Bcl-2 was significantly decreased, and there were significant between-group differences (allP<0.05). After the treatment, there were statistically significant differences among the treatment groups in the neurological function impairment score, cerebral infarct area ratio and apoptosis in the ischemic side of rats, as well as the expressions of Bcl-2, Bax and Caspase-3 (allP<0.05), and from the figures, tables and statistical analysis, it was found that a better tendency in the acupuncture plus mild hypothermia group than the acupuncture group or mild hypothermia group.Conclusion: Acupuncture plus mild hypothermia can protect the brain cells by improving neurological function impairment, decreasing cerebral infarct area ratio, reducing the number of apoptotic cells in the ischemic area and regulating the expressions of apoptosis related proteins to inhibit apoptosis.