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.

Protective effect of NLRP12 knockdown on retinal damage in rats with ocular hypertension and its mechanism of inhibiting cell pyroptosis / 中华实验眼科杂志

Objective:To investigate the effect and mechanism of NOD-like receptor family pyrin domain containing 12 (NLRP12) knockdown on inflammatory factor levels and retinal injury in retinal ganglion cells (RGCs) of rats with high intraocular pressure.Methods:Seventy SPF adult male SD rats were selected and randomized into control group, high intraocular pressure (IOP) group, high IOP+ small interfering RNA negative control (siNC) group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ recombinant rat caspase-1 (rrcaspase-1) group, with 14 rats in each group.Rats in the control group were only treated with conjunctival incision in the right eye, and ocular hypertension model was established in the other four groups with external scleral vein cauterization.High IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group were injected with siNC, siNLRP12 and siNLRP12+ rrcaspase-1 reagent via the tail vein, respectively.The IOP of the right eye was measured at 1 day, 1, 2 and 3 weeks after the operation.Three weeks after the operation, the retinal structure was observed by hematoxylin-eosin staining, and the number of RGCs in each group was counted.RGCs were divided into control group, rrcaspase-1 group, siNC+ rrcaspase-1 group, siNLRP12+ rrcaspase-1 group.The cells in rrcaspase-1 group, siNC+ rrcaspase-1 group and siNLRP12+ rrcaspase-1 group were treated with rrcaspase-1, siNC+ rrcaspase-1 and siNLRP12+ rrcaspase-1 reagent for 24 hours, respectively.No treatment was given to the control group.The expression levels of NLRP12, caspase-1 and cleaved-caspase-1 proteins in RGCs and retinal tissue were detected by Western blot.The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in rat serum or cell culture supernatant were detected by enzyme-linked immunosorbent assay.The study protocol was approved by the Animal Ethics Committee of the First People's Hospital of Chenzhou (No.2020086).Results:Compared with control group, the IOP was higher in high IOP group at 1, 2 and 3 weeks after cauterization, and the differences were statistically significant (all at P<0.05). The retinal tissue was clear with the RGCs in a single layer arrangement in the control group.In the high IOP group and the high IOP+ siNC group, the RGCs layer was loose and the inner plexiform layer was thin.The inner plexiform layer was thickened in high IOP+ siNLRP12 group compared with high IOP group, and the RGCs layer was loose in the high IOP+ siNLRP12 group and the high IOP+ siNLRP12+ rrcaspase-1 group.The number of RGCs in control group, high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group was 119.31±23.25, 89.19±16.98, 88.87±13.92, 109.33±10.25 and 92.89±12.58, respectively, showing a statistically significant overall difference ( F=201.932, P<0.001). The number of RGCs was lower in the high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group than the control group, higher in the high IOP+ siNLRP12 group than the high IOP+ siNC group, and lower in the high IOP+ siNLRP12+ rrcaspase-1 group than the high IOP+ siNLRP12 group, and the differences were statistically significant (all at P<0.05). The relative expressions of caspase-1 and cleaved-caspase-1 proteins and the concentrations of TNF-α and IL-1β in the retinal tissue were higher in high IOP group, high IOP+ siNC group, high IOP+ siNLRP12 group and high IOP+ siNLRP12+ rrcaspase-1 group than control group, higher in high IOP+ siNLRP12 group than high IOP+ siNC group, and higher in high IOP+ siNLRP12+ rrcaspase-1 group than high IOP+ siNLRP12 group (all at P<0.05). Relative expression levels of caspase-1 and cleaved-caspase-1 protein were increased in rrcaspase-1 group and siNC+ rrcaspase-1 group compared with control group, and relative expression levels of NLRP12, caspase-1 and cleaved-caspase-1 protein were decreased in siNLRP12+ rrcaspase-1 group compared with control group (all at P<0.05). The relative mass concentrations of TNF-α and IL-1β were increased in rrcaspase-1 group, siNC+ rrcaspase-1 group and siNLRP12+ rrcaspase-1 group compared with the control group (all at P<0.05). Relative expression levels of NLRP12, caspase-1 and cleaved-caspase-1 proteins and relative mass concentrations of TNF-α and IL-1β in siNLRP12+ rrcaspase-1 group were lower than those in siNC+ rrcaspase-1 group (all at P<0.05). Conclusions:Knockdown of NLRP12 can reduce the inflammatory response and retinal injury induced by high IOP by inhibiting the activation of caspase-1.

Babaodan Alleviates APAP-induced Acute Liver Injury in Mice by Inhibiting NLRP3/Caspase-1 Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effect of Babaodan （BBD） on the NOD-like receptor pyrin domain containing 3/cysteine aspartate-specific protease-3 （NLRP3/Caspase-1） pathway proteins in mice with acetaminophen （APAP）-induced acute liver injury. MethodC57BL/6 mice were randomly grouped， and BBD （75， 150， 300 mg·kg-1， ig） was administered twice a day for three days. After 2 hours of the last administration， the mice were treated with APAP （400 mg·kg-1， ip）， and the eyeballs were removed to collect blood after 14 hours. Then they were sacrificed by cervical dislocation for sample collection. Hematoxylin-eosin （HE） staining was used to observe the morphological changes of liver tissue cells， and biochemical methods were used to detect the activities of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， superoxide dismutase （SOD）， malondialdehyde （MDA） and myeloperoxidase （MPO） in serum of mice in each group. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was performed to determine the mRNA expression of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β） and IL-6， and Western blot was performed to determine the protein expression of cyclooxygenase-2 （COX-2）， inducible nitric oxide synthase （iNOS）， NLRP3， Caspase-1 and IL-18 in the liver of mice. ResultCompared with the conditions in normal group， the hepatic lobule structure of mice in the model group was partially destroyed， and the hepatic sinusoids were dilated. And the expression levels of ALT and AST in serum， the protein levels of NLRP3， Caspase-1， iNOS， IL-18 and COX-2 and the mRNA levels of IL-1β， IL-6 and TNF-α were increased （P<0.05， P<0.01）. Compared with the model group， the administration groups had improvement in liver cell rupture and hepatic sinusoidal compression， and a dose-dependent decrease in the levels of ALT and AST in serum as well as the protein levels of NLRP3， Caspase-1， iNOS， IL-18 and COX-2 and the the mRNA levels of IL-1β， IL-6 and TNF-α in liver tissue （P<0.05， P<0.01）. ConclusionBBD can reduce APAP-induced acute liver injury in mice. The mechanism may be related to anti-oxidative stress， inhibition of NLRP3/Caspase-1 pathway， and decreased expression levels of IL-1β， IL-18， TNF-α and IL-6.

Effect of multi-glycosides of Tripterygium wilfordii on renal injury in diabetic kidney disease rats through NLRP3/caspase-1/GSDMD pyroptosis pathway / 中国中药杂志

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.

Effect of procalcitonin on lipopolysaccharide-induced expression of nucleotide-binding oligomerization domain-like receptor protein 3 and caspase-1 in human umbilical vein endothelial cells / 中国当代儿科杂志

OBJECTIVES@#To study the effect of procalcitonin (PCT) on lipopolysaccharide (LPS)-induced expression of the pyroptosis-related proteins nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and caspase-1 in human umbilical vein endothelial cells (HUVECs).@*METHODS@#HUVECs were induced by LPS to establish a model of sepsis-induced inflammatory endothelial cell injury. The experiment was divided into two parts. In the first part, HUVECs were randomly divided into four groups: normal control, LPS (1 μg/mL), PCT (10 ng/mL), and LPS+PCT (n=3 each). In the second part, HUVECs were randomly grouped: normal control, LPS, and LPS+PCT of different concentrations (0.1, 1, 10, and 100 ng/mL) (n=3 each). Quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of NLRP3 and caspase-1 in each group.@*RESULTS@#In the first experiment: compared with the normal control group, the PCT, LPS, and LPS+PCT groups had significantly upregulated mRNA and protein expression levels of NLRP3 and caspase-1 (P<0.05); compared with the LPS group, the LPS+PCT group had significantly downregulated mRNA and protein expression levels of NLRP3 and caspase-1 (P<0.05). In the second experiment: compared with those in the LPS group, the mRNA and protein expression levels of NLRP3 and caspase-1 in the LPS+PCT of different concentrations groups were significantly downregulated in a concentration-dependent manner (P<0.05).@*CONCLUSIONS@#LPS can promote the expression of the pyroptosis-related proteins NLRP3 and caspase-1 in HUVECs, while PCT can inhibit the LPS-induced expression of the pyroptosis-related proteins NLRP3 and caspase-1 in HUVECs in a concentration-dependent manner.

Experimental study of cardioprotective effects of Cinnamomi Ramulus and Cinnamomi Cortex formula granules on myocardial ischemia/reperfusion injury in rats based on efficacy of "warming and coordinating heart Yang" / 中国中药杂志

This study aimed to parallelly investigate the cardioprotective activity of Cinnamomi Ramulus formula granules(CRFG) and Cinnamomi Cortex formula granules(CCFG) against acute myocardial ischemia/reperfusion injury(MI/RI) and the underlying mechanism based on the efficacy of "warming and coordinating the heart Yang". Ninety male SD rats were randomly divided into a sham group, a model group, CRFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, and CCFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, with 15 rats in each group. The sham group and the model group were given equal volumes of normal saline by gavage. Before modeling, the drug was given by gavage once a day for 7 consecutive days. One hour after the last administration, the MI/RI rat model was established by ligating the left anterior descending artery(LAD) for 30 min ischemia followed by 2 h reperfusion except the sham group. The sham group underwent the same procedures without LAD ligation. Heart function, cardiac infarct size, cardiac patho-logy, cardiomyocyte apoptosis, cardiac injury enzymes, and inflammatory cytokines were determined to assess the protective effects of CRFG and CCFG against MI/RI. The gene expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain protein 3(NLRP3) inflammasome, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate specific proteinase-1(caspase-1), Gasdermin-D(GSDMD), interleukin-1β(IL-1β), and interleukin-18(IL-18) were determined by real-time quantitative polymerase chain reaction(RT-PCR). The protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD were determined by Western blot. The results showed that both CRFG and CCFG pretreatments significantly improved cardiac function, decreased the cardiac infarct size, inhibited cardiomyocyte apoptosis, and reduced the content of lactic dehydrogenase(LDH), creatine kinase MB isoenzyme(CK-MB), aspartate transaminase(AST), and cardiac troponin Ⅰ(cTnⅠ). In addition, CRFG and CCFG pretreatments significantly decreased the levels of IL-1β, IL-6, and tumor necrosis factor-α(TNF-α) in serum. RT-PCR results showed that CRFG and CCFG pretreatment down-regulated the mRNA expression levels of NLRP3, caspase-1, ASC, and downstream pyroptosis-related effector substances including GSDMD, IL-18, and IL-1β in cardiac tissues. Western blot revealed that CRFG and CCFG pretreatments significantly decreased the protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD in cardiac tissues. In conclusion, CRFG and CCFG pretreatments have obvious cardioprotective effects on MI/RI in rats, and the under-lying mechanism may be related to the inhibition of NLRP3/caspase-1/GSDMD signaling pathway to reduce the cardiac inflammatory response.

Effects of Huangqin Tang on NLRP3/Caspase-1 pathway in mice model of ulcerative colitis / 中国中药杂志

The aim of this study was to explore the effects of Huangqin Tang(HQT) on the NLRP3/Caspase-1 signaling pathway in mice with DSS-induced ulcerative colitis(UC). C57BL/6J mice were randomly divided into a blank group, a model group(DSS group), and low-, medium-and high-dose HQT groups(HQT-L, HQT-M, and HQT-H), and western medicine mesalazine group(western medicine group). The UC model was induced in mice. Subsequently, the mice in the HQT-L, HQT-M, HQT-H groups, and the western medicine group were given low-, medium-, high-dose HQT, and mesalazine suspension by gavage, respectively, while those in the blank and DSS groups were given an equal volume of distilled water by gavage. After 10 days of administration, the body weight, DAI scores, and colonic histopathological score of mice in each group were determined. The levels of IL-6, IL-10, IL-1β, and TNF-α in serum were determined by ELISA. The mRNA expression of NLRP3 and Caspase-1 in colon tissues was determined by RT-qPCR. The protein expression of NLRP3 and Caspase-1 in colon tissues was detected by immunohistochemistry. The results showed that compared with the blank group, the DSS group showed decreased body weight of mice and increased DAI scores and intestinal histopathological score. Compared with the DSS group, the HQT groups and the western medicine group showed improved DAI scores, especially in the HQT-M, HQT-H, and the western medicine groups(P<0.05). The intestinal histopathological scores of the HQT groups and the western medicine group significantly decreased, especially in the HQT-M, HQT-H, and the western medicine groups(P<0.05). In addition, compared with the blank group, the DSS group showed elevated expression of NLRP3 and Caspase-1 in colon tissues, increased serum levels of IL-6, IL-1β, and TNF-α, and decreased IL-10 level. Compared with the DSS group, the HQT groups and the western medicine group displayed decreased expression of NLRP3 and Caspase-1 in colon tissues, reduced serum levels of IL-6, IL-1β, and TNF-α, and increased IL-10 level. The improvement was the most significant in the HQT-H group and the western medicine group(P<0.01). In conclusion, HQT may reduce the expression of NLRP3 and Caspase-1 in colon tissues, reduce the se-rum levels of IL-6, IL-1β, and TNF-α, and increase the expression of IL-10 by regulating the classic pyroptosis pathway of NLRP3/Caspase-1, thereby improving the symptoms of intestinal injury and inflammatory infiltration of intestinal mucosa in DSS mice to achieve its therapeutic effect.

Puerarin protects renal ischemia-reperfusion injury in rats through NLRP3/Caspase-1/GSDMD pathway

Purpose: To observe the effect of puerarin on renal ischemia-reperfusion (I/R) injury in rats, and to explore its mechanism based on NLRP3/Caspase-1/GSDMD pathway. Methods: Twenty-one Sprague-Dawley rats were divided into three groups: sham-operated group (sham), model group (RIRI), and puerarin treatment group (RIRI + Pue). The model of acute renal I/R injury was established by cutting the right kidney and clamping the left renal pedicle for 45 min. Results: Renal function parameters were statistically significant in group comparisons. The renal tissue structure of rats in sham group was basically normal. Pathological changes were observed in the RIRI group. The renal pathological damage score and apoptosis rate in the RIRI group were higher than those in the sham group, and significantly lower in the RIRI + Pue group than in the RIRI group. Indicators of oxidative stress-superoxide dismutase, malondialdehyde, and glutathione peroxidase-were statistically significant in group comparisons. Compared with the sham group, the relative expressions of NLRP3, Caspase-1 and GSDMD proteins in the RIRI group were increased. Compared with the RIRI group, the RIRI + Pue group had significant reductions. Conclusions: Puerarin can inhibit the activation of NLRP3/Caspase-1/GSDMD pathway, inhibit inflammatory response and pyroptosis, and enhance the antioxidant capacity of kidney, thereby protecting renal I/R injury in rats.

Jiedu Huoxue Prescription Regulates NF-κB/NLRP3/Caspase-1 to Promote Vascular Reendothelialization in Rat Model of Injured Thoracic Aorta / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Jiedu Huoxue prescription in promoting the reendothelialization of injured vessels by regulating the nuclear factor （NF）-κB/NOD-like receptor protein 3 （NLRP3）/cysteine-aspartic acid protease （Caspase）-1-mediated pyroptosis. MethodA rat model of injured thoracic aorta was established by balloon injury， and 36 rats were assigned into shame surgery， model， low-， medium-， and high-dose Jiedu Huoxue prescription， and atorvastatin calcium tablet groups. The injured aortic segment was collected 28 days after surgery. Hematoxylin-eosin （HE） staining and Evans blue staining were conducted to reveal the changes of vascular structural morphology and the reendothelialization of blood vessels， respectively. The enzyme-linked immunosorbent assay （ELISA） was employed to determine the levels of tumor necrosis factor-α （TNF-α）， intercellular adhesion molecule-1 （ICAM-1）， interleukin （IL）-1β， and nitric oxide （NO） in the serum. Western blotting was employed to determine the expression of endothelial nitric oxide synthase （eNOS）， NF-κB p65， phospho-NF-κB p65 （p-NF-κB p65）， NLRP3， and Caspase-1 in the vascular tissue. ResultThe model group showed thickened endovascular membrane， proliferation and disarrangement of smooth muscle cells of the artery wall， obvious inflammatory cell infiltration， and narrowed luminal area. Jiedu Huoxue prescription and atorvastatin calcium tablets mitigated the pathological changes of the thoracic aorta in different degrees. After balloon injury， the endothelial coverage rate of the model group decreased significantly， while Jiedu Huoxue prescription and atorvastatin calcium tablets increased the reendothelialization rate （P<0.05）. Compared with the shame surgery group， the model group showed elevated levels of TNF-α， ICAM-1， and IL-1β （P<0.01） and lowered NO level （P<0.01） in the serum. In addition， the model group presented down-regulated protein level of eNOS （P<0.01） and up-regulated phosphorylation of pyroptosis-associated proteins NLPR3， Caspase-1， and NF-κB p65 in the vascular tissue （P<0.05， P<0.01）. Compared with the model group， Jiedu Huoxue prescription and atorvastatin calcium tablets lowered TNF-α， ICAM-1， and IL-1β levels （P<0.05， P<0.01） and elevated the NO level in the serum （P<0.05， P<0.01）. Moreover， the drugs up-regulated the expression of eNOS （P<0.01） and down-regulated the expression of NLRP3， Caspase-1， and NF-κB p65 （P<0.05， P<0.01） in the vascular tissue. ConclusionJiedu Huoxue prescription can promote the reendothelialization and inhibit the intimal hyperplasia of vessels after balloon injury by regulating the NF-κB/NLRP3/Caspase-1 pathway to inhibit pyroptosis and reduce endothelial inflammatory injury.

Mechanism of Shenqi Yiliu Prescription Combined with Cisplatin on H22 Liver Cancer-bearing Mice Based on NLRP3/Caspase-1/GSDMD Pyroptosis Pathway / 中国实验方剂学杂志

ObjectiveTo explore the anti-tumor effect and mechanism of Shenqi Yiliu prescription in the intervention of pyroptosis. MethodTen male BALB/c mice were randomly selected and assigned to the blank group. The remaining 40 mice underwent the induction of the liver cancer xenograft model. After 5 days of modeling， 40 surviving mice were randomly divided into model group， cisplatin group ［2.5×10-3 g·kg-1·（3 d）-1］， Shenqi Yiliu prescription group （27 g·kg-1·d-1）， and a combination group （Shenqi Yiliu prescription group + cisplatin）. The mice in the blank group and the model group were treated with an equal volume of normal saline for 10 days. The general conditions of mice in each group were observed. After the intervention， the tumor weight of the mice was weighed and the tumor inhibition rate was calculated. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in tumor tissues. The levels of mouse liver function indicators， including alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were detected. The TdT-mediated dUTP-biotin nick end labeling （TUNEL） assay was used to detect DNA damage in mouse tumor tissue cells. Immunohistochemistry （IHC）， immunofluorescence （IF）， and Western blot were used to detect the protein expression levels of NOD-like receptor protein 3 （NLRP3）， cysteinyl aspartate-specific protease-1 （Caspase-1）， and gasdermin D （GSDMD） in tumor tissues. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in tumor tissues were detected by enzyme-linked immunosorbent assay （ELISA）. ResultCompared with the mice in the blank group， those in the model group were in a poor mental state， sleepy， and lazy， and their fur color was dull， with increased levels of serum ALT and AST in liver function tests （P<0.01）. Compared with the model group， the groups with drug intervention showed improved mental state， inhibited tumor growth to varying degrees， and decreased tumor weight， and the tumor inhibition rate in the combination group was the highest （P<0.01）. HE staining showed that the pathological and morphological lesions of the tumor tissues in the model group were significant， while those in all groups with drug intervention were improved to a certain extent. The karyolysis and nuclear rupture in the Shenqi Yiliu prescription group and the combination group were more significant. In the liver function test， the serum ALT and AST levels of mice in the Shenqi Yiliu prescription group and the combination group decreased （P<0.01）， and the inflammatory factors IL-1β and IL-18 in each group with drug intervention decreased （P<0.05， P<0.01）. Among them， the declining trend of IL-1β and IL-18 in the Shenqi Yiliu prescription group was the most significant （P<0.01）. TUNEL staining showed that the positive TUNEL staining in each group with drug intervention decreased after intervention （P<0.05， P<0.01）， especially the cisplatin group and Shenqi Yiliu prescription group （P<0.01）. Western blot， IHC， and IF found that the protein expression levels of NLRP3， Caspase-1， and GSDMD in each group with drug intervention decreased （P<0.05， P<0.01）. Compared with the mice in the cisplatin group， those in the Shenqi Yiliu prescription group and the combination group had better mental state and regular tumor morphology， and the tumor weight of the mice in the combination group decreased （P<0.05）. The levels of ALT and AST in the Shenqi Yiliu prescription group decreased （P<0.05）， and the levels of IL-1β and IL-18 in the Shenqi Yiliu prescription group and the combination group decreased （P<0.05， P<0.01）， especially in the combination group （P<0.01）. The results of IHC showed that the expression of GSDMD protein in the tumor tissues of mice in the combination group was reduced （P<0.01）. IF detection showed that the expression of NLRP3 in the tumor tissues of the Shenqi Yiliu prescription group was reduced （P<0.01）. The results of Western blot showed that the expression level of NLRP3 protein in the Shenqi Yiliu prescription group and the combination group decreased （P<0.01）， and the expression level of Caspase-1 protein in the combination group decreased （P<0.01）. The decrease in GSDMD protein expression was not significant， and the difference was not statistically significant. ConclusionShenqi Yiliu prescription combined with cisplatin has an obvious anti-tumor effect， which may be achieved by down-regulating the NLRP3/Caspase-1/GSDMD inflammatory pyroptosis pathway to inhibit cell pyroptosis， and relieve the inflammatory response in mice with liver cancer.

AIM2 inflammasome activation may mediate high mobility group box 1 release in murine allergic rhinitis / Ativação do inflamassoma AIM2 pode mediar a liberação da proteína do grupo Box-1 de alta mobilidade no modelo murino de rinite alérgica

Abstract Introduction: High mobility group box 1 protein participates in the pathogenesis of allergic rhinitis. Activation of the inflammasome can mediate the release of high mobility group box 1. The role of the absent in melanoma 2 inflammasome in allergic rhinitis remains unclear. Objective: This study aimed to investigate the function of absent in melanoma 2 inflammasome in murine allergic rhinitis and the interaction between high mobility group box 1 and the absent in melanoma 2 inflammasome. Methods: A murine allergic rhinitis model was established using twenty Balb/c mice. Expression of the components of the absent in melanoma 2 inflammasome: absent in melanoma 2, apoptosis-associated speck-like protein containing a CARD (Asc), caspase-1 p20, and additional nod-like receptor family pyrin domain containing 3 (Nlrp3) were detected by western blotting during allergic rhinitis. Alterations of absent in melanoma 2, caspase-1, and high mobility group box 1 after ovalbumin challenge were demonstrated by immunohistochemistry. TdT-mediated dUTP Nick end labeling, TUNEL assay, and cleavage of caspase-3 and PARP-1 were used for the observation of pyroptosis. Results: Eosinophilia and goblet cell infiltration were observed in the nasal mucosa of mice in the allergic rhinitis group. Absent in melanoma 2, Asc, and caspase-1 p20 increased after ovalbumin exposure while Nlrp3 did not. High mobility group box 1 was released in the nasal mucosa of allergic rhinitis mice. TUNEL-positive cells increased in the epithelium and laminae propria, whereas cleavage of caspase-3 and PARP-1 was not observed. Conclusions: The absent in melanoma 2 inflammasome was activated and pyroptosis may occur in the nasal mucosa after ovalbumin treatment. These may contribute to the translocation of high mobility group box 1 and the development of allergic rhinitis.

Resumo Introdução: A proteína do grupo Box-1 de alta mobilidade participa da patogênese da rinite alérgica. A ativação do inflamassoma pode mediar a liberação de proteína do grupo Box-1 de alta mobilidade. O papel do inflamassoma ausente no melanoma 2 na rinite alérgica permanece incerto. Objetivo: Investigar a função do inflamassoma ausente no melanoma 2 em um modelo murino de rinite alérgica e a interação entre a proteína do grupo Box-1 de alta mobilidade e o inflamassoma ausente no melanoma 2. Método: Um modelo murino de rinite alérgica foi estabelecido com 20 camundongos Balb/c. A expressão dos componentes do inflamassoma ausente no melanoma 2, da proteína speck-like associada à apoptose com CARD (Asc), da caspase-1 p20 e do domínio de pirina da família NLR adicional com 3 (Nlrp3) foi detectada por western blotting durante a rinite alérgica. Alterações de inflamassoma ausente no melanoma 2, na caspase-1 e na proteína do grupo Box-1 de alta mobilidade após o teste de provocação com ovalbumina foram demonstradas por imuno-histoquímica. O ensaio dUTP Nick-End Labeling mediado por TdT, TUNEL e clivagem de caspase-3 e PARP-1 foram usados para a observação de piroptose. Resultados: Eosinofilia e infiltração de células caliciformes foram observadas na mucosa nasal de camundongos do grupo rinite alérgica. Inflamassoma ausente no melanoma 2, Asc e caspase-1 p20 aumentou após a exposição à ovalbumina, enquanto Nlrp3 não aumentou. A proteína do grupo Box-1 de alta mobilidade foi liberada na mucosa nasal de camundongos com rinite alérgica. As células TUNEL-positivas aumentaram no epitélio e na lâmina própria, enquanto a clivagem da caspase-3 e a PARP-1 não foram observadas. Conclusão: O inflamassoma ausente no melanoma 2 foi ativado e pode ocorrer piroptose na mucosa nasal após o tratamento com ovalbumina. Esses fatores podem contribuir para a translocação de proteína do grupo Box-1 de alta mobilidade e o desenvolvimento de rinite alérgica.

The role of Caspase-1/GSDMD-mediated cell pyroptosis in anti-tumor effect of cisplatin in triple-negative breast cancer / 中华内分泌外科杂志

Objective:To investigate the role of Caspase-1/gasdermin D (GSDMD) -mediated cell pyroptosis in anti-tumor effect of cisplatin (DDP) in triple-negative breast cancer (TNBC) .Methods:HE staining and immunohistochemical staining were performed to detect the morphological changes and the expression of pyroptosis/apoptosis pathway related proteins in TNBC tissues before and after DDP-based neoadjuvant chemotherapy (NACT) . The TNBC cell line MDA-MB-231 was treated with DDP and the morphological changes were observed. The type of cell death induced by DDP was analyzed by Annexin V-FITC/PI double staining and flow cytometry. Lactate dehydrogenase (LDH) release assay and ELISA were performed to detect the release of LDH and inflammatory factors (IL-18 and IL-1β) in cell culture supernatant after DDP treatment. Western blot (WB) was performed to detect the expression of pyroptosis/apoptosis pathway related proteins in cells after DDP treatment. MDA-MB-231 cells treated with DDP were co-treated with caspase-1 specific inhibitor to inhibit pyroptois or co-treated with caspase-3 specific inhibitor to inhibit apoptosis. The effect of caspase-1 inhibitor or caspase-3 inhibitor on the anti-tumor effect of DDP was detected by MTT assay, clone formation assay, transwell assay and would healing test.Results:Reactive changes in the breast surgical specimen after DDP-based NACT included cell swelling and inflammatory cell aggregation around the tumor bed, which were more similar to pyroptosis. The up-regulation of key molecules of pyroptosis pathway post-NACT was significantly higher than that of key molecules of apoptosis pathway. Further experiments in vitro showed that DDP could induce MDA-MB-231 cells to show pyroptosis-like changes characterized by large bubbles blowing from the cellular membrane. Flow-cytometry analyses showed that the death type of MDA-MB-231 cells caused by DDP was mainly Annexin V +PI + cells (mainly lytic cells, such as pyroptosis) . Additionally, DDP treatment induced significant activation of caspase-1 and GSDMD, increased the release of LDH, IL-18 and IL-1β, however, the activation level of caspase-3, which dominates the apoptosis pathway, was significantly lower than that of caspase-1/GSDMD. Moreover, caspase-1 inhibitors (blocking the classical pyroptosis pathway) had a significantly greater inhibitory effect on the anti-tumor effect of DDP than caspase-3 inhibitors (blocking the apoptosis pathway) . Conclusion:Caspase-1/GSDMD mediated pyroptosis may play a leading role in the anti-tumor effect of DDP in triple-negative breast cancer.

The role of TSG-6 in inhibiting the secretion of IL-1β by bone-marrow-derived macrophages / 中国职业医学

@#Objective To investigate the role of tumor necrosis factor alpha stimulated gene/inducible protein 6（TSG-6）on free silica（SiO2 ）-induced secretion of pro-inflammatory cytokine interleukin（IL）-1β by bone-marrow-derived macrophages （BMDMs）. Methods i）BMDMs isolated from bone marrow were divided into eight groups：the control group was untreated； lipopolysaccharide （LPS） group was stimulated by LPS with a concentration of 50 µg/L；The TSG-6 control group was stimulated by 100 µg/L of recombinant mouse TSG-6. SiO2 model group was pre-stimulated with LPS for four hours，and then stimulated with SiO2 suspension at a final concentration of 250 mg/L；Different dose of TSG-6 groups were firstly treated with above concentrations of LPS and SiO2 suspension，then 10，20，50 and 100 µg/L of recombinant mouse TSG-6 were added respectively；After 16 hours of culture，the cells were collected and the level of IL-1β in BMDMs supernatant was detected by enzyme-linked immunosorbent assay（ELISA）to screen optimal concentration of TSG-6. ii）The cells of the control group，LPS group，SiO2 model group，TSG-6 optimal concentration group and TSG-6 control group were collected. The expression of IL-1β and components of its related pathways in BMDMs was detected by Western blot，including IL-1β，pro-IL-1β，caspase-1，pro dcaspase-1，asc type amino acid transport and NOD-like receptor protein 3（NLRP3）. Results i）Compared with the control group，the expression of IL-1β in SiO2 model group was increased significantly（P<0.01）. Compared with SiO2 model group，the expression of IL-1β in 20，50，100 µg/L dose of TSG-6 groups were decreased significantly（all P<0.01），and the optimal concentration of TSG-6 was found to be 100 µg/L. ii）Compared with the control group and LPS group，the relative expression levels of IL-1β，caspase-1 and NLRP3 in SiO2 model group were increased significantly （all P<0.05）. Compared with SiO2 model group，the expression levels of IL-1β、caspase-1 and NLRP3 were decreased in 100 µg/L TSG-6 group（all P<0.05）. Conclusion TSG-6 could inhibit BMDMs to secret pro-inflammatory cytokine IL-1β by down-regulating the expression of NLRP3 and caspase-1.

Effect of Banxia Xiexintang on NLRP3/Caspase-1 Pyroptosis Pathway in Rats with Ulcerative Colitis / 中国实验方剂学杂志

ObjectiveTo explore the effect of Banxia Xiexintang （BXT） on the NOD-like receptor protein 3 （NLRP3）/cysteinyl aspartate-specific protease-1 （Caspase-1） pyroptosis pathway and its downstream factors in rats with ulcerative colitis （UC）， and to explain the mechanism of BXT in the treatment of UC. MethodSD rats were randomly divided into normal control group， model group， low- and high-dose BXT groups （6.3， 12.6 g·kg-1·d-1）， and salazosulfapyridine （SASP） group （0.42 g·kg-1·d-1）， with 7 rats in each group. The UC model was induced by intragastric administration of 2.5% dextran sodium sulfate （DSS） solution for 10 days， followed by drug intervention for 7 days. The general state of rats was observed during the experiment， and the disease activity index （DAI） score was calculated during the administration period. At the end of the experiment， colonic tissues were collected for hematoxylin-eosin （HE） staining to observe the pathological changes and the curative effect of BXT. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA transcriptional levels of NLRP3， Caspase-1， gasdermin D （GSDMD）， and interleukin （IL）-1β in colonic tissues. Western blot was used to detect the protein expression of NLRP3， Caspase-1， GSDMD， and IL-1β in colonic tissues to explore the therapeutic mechanism of BXT. ResultCompared with the normal control group， the model group showed increased DAI score， pathological changes in colonic tissues， and up-regulated mRNA and protein expression of NLRP3， Caspase-1， GSDMD， and IL-1β （P<0.05， P<0.01）. Compared with the model group， the groups with drug intervention showed reduced DAI scores and improved pathological changes in colonic tissues. The mRNA and protein expression levels of NLRP3， Caspase-1， GSDMD， and IL-1β in colonic tissues of the BXT groups were significantly down-regulated or tended to be down-regulated， especially in the low-dose BXT group （P<0.05， P<0.01）. ConclusionBXT can inhibit pyroptosis and alleviate inflammation in rats with UC by regulating the NLRP3/Caspase-1 pathway.

Mechanism of Tingli Dazao Xiefeitang on Cell Pyrolysis and Apoptosis Induced by Caspase-1 in A549 Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of coking death and apoptosis of A549 cells induced by Tingli Dazao Xiefeitang. MethodA549 cells were randomized into blank group， traditional Chinese medicine（TCM） low， medium， and high concentration groups， which were treated with 20， 40， 60 mg·L-1 Tingli Dazao Xiefeitang， and TCM low， medium， and high concentration groups， respectively， and blank group was treated with equal volume culture medium. After 48 h of treatment， cell migration was detected by scratch assay and cell apoptosis was detected by flow cytometry. The relative expression levels of cysteine aspartate protease-1（Caspase-1）， NOD-like receptor protein 3 （NLRP3）， dermoderin D （GSDMD）， Survivin protein and nuclear transcription factor -κB （NF-κB） pathway proteins were detected by Western blot. The levels of intracellular reactive oxygen species （ROS） were determined by DCFH-DA fluorescence probe， and the contents of tumor necrosis factor -β （TNF-β） and interleukin-1β （IL-1β） in supernatant were determined by enzyme-linked immunosorbent assay （ELISA）. ResultCompared with blank group， the scratch healing rate， apoptosis rate， relative expression of Survivin protein， Caspase-1， GSDMD， NLRP3， ROS and NF-κB phosphorylation levels were significantly increased in low， medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. Compared with the low concentration group， the scratch healing rate， apoptosis rate， Survivin protein relative expression， Caspase-1， GSDMD， NLRP3 relative expression， ROS and NF-κB phosphorylation levels were significantly increased in the medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. Compared with the TCM group， the scratch healing rate， apoptosis rate， Survivin protein relative expression， Caspase-1， GSDMD， NLRP3 relative expression， ROS and NF-κB phosphorylation levels were significantly increased in the high concentration group. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. ConclusionTingli Dazao Xiefeitang can improve NLRP3 protein expression， inhibit Survivin protein expression and promote apoptosis of A549 cells. At the same time， it can activate NF-κB pathway and ROS system， up-regulate the expression of Caspase-1 and GSDMD， mediate scortosis of A549 cells.

Total Flavonoids of Peony Flower Regulate NLRP3 Inflammasome and Expression of Inflammatory Cytokines in Gouty Nephropathy Rats / 中国实验方剂学杂志

ObjectiveTo explore the possible mechanism of total flavonoids of peony flower （TFPF） in protecting rats from gouty nephropathy and provide data support for the pharmaceutical research on the treatment of gouty nephropathy. MethodGouty nephropathy rat model was established by adenine combined with ethambutol. Rats were randomly assigned into blank control group， model group， allopurinol （42 mg·kg-1） group， Tongfengshu tablets （600 mg·kg-1， positive control） group， and TFPF （260， 130， and 65 mg·kg-1） groups. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of tumor necrosis factor-α （TNF-α）， monocyte chemoattractant protein-1 （MCP-1）， interleukin-1β （IL-1β）， and interleukin-18 （IL-18） in rat serum and those of transforming growth factor-β1 （TGF-β1） and IL-1β in renal homogenate. Hematoxylin-eosin（HE） staining was carried out for observation of the morphological changes of renal cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） was conducted for observation of the DNA damage in renal cells. The expression of NOD-like receptor protein 3 （NLRP3）， cysteine aspartic acid protease（Caspase）-1 and IL-1β were observed by immunohistochemistry. The expression levels of NLRP3， Caspase-1 and nuclear transcription factor -κB （NF-κB） in renal tissues were detected by Western blot. ResultCompared with blank group， the contents of TNF-α， MCP-1， IL-1β， IL-18， and TGF-β1 in serum of model group were significantly increased （P<0.01）， and the expressions of NLRP3， Caspase-1， NF-κB and IL-1β in kidney of model group were significantly increased （P<0.01）. The renal tissue cells showed cytoplasmic swelling， cell membrane rupture， and the number of nuclear pyknotic fracture increased. The positive rate of TUNEL staining was significantly increased in model group （P<0.01）， and the contents of IL-1β and TGF-β1 in renal tissue homogenate were significantly increased （P<0.01）. Compared with model group， the contents of inflammatory factors TNF-α， IL-1β and IL-18 in serum of rats in TFPF high- and medium-dose groups could be decreased to different degrees （P<0.05， P<0.01）， while the content of MCP-1 in TFPF high-dose group was significantly decreased （P<0.01）. The content of TGF-β1 in renal tissue homogenate in TFPF high- and medium-dose groups was significantly decreased （P<0.05， P<0.01）， and the content of IL-1β in renal tissue homogenate in TFPF medium-dose group was significantly decreased （P<0.01）. HE staining showed that each dose group of TFPF could improve the status of renal tubular epithelial cells， reduce cytoplasmic swelling and the number of nuclear pyknosis to varying degrees. The positive rate of TUNEL staining was decreased （P<0.01） and DNA damage was decreased. The expression of NLRP3， Caspase-1， IL-1β and NF-κB protein in renal tissue cells was inhibited （P<0.05， P<0.01）. ConclusionTFPF protects rats from gouty nephropathy by inhibiting the secretion of inflammatory cytokines. Specifically， it may inhibit the activation of NF-κB and NLRP3/Caspase-1 pathways to reduce the expression， maturation， and release of inflammatory cytokines such as IL-1β and IL-18 and further inhibit pyroptosis， thereby reversing the inflammatory injury of kidney in gouty nephropathy.

Effects of electroacupuncture at Zusanli on duodenal Caspase-1 and GSDMD of rats with functional dyspepsia / 中华物理医学与康复杂志

Objective:To explore any effect of electroacupuncture (EA) at the Zusanli point on the scorch death of duodenal cells in rats with functional dyspepsia (FD) and possible mechanisms.Methods:Twenty-four 7-day-old Sprague-Dawley rats were randomly divided into a blank group, a model group and an EA group, each of 8. FD was induced in both the model and EA group rats using iodoacetamide gavage with tail-clip stimulation. After successful modeling the EA group was given acupuncture at the Zusanli point and then connected with a Korean acupuncture point nerve stimulator for 2 weeks. The other 2 groups were not given any intervention. The rats′ body weight was recorded before and after the modeling, as well as 7 and 14 days later. The gastric emptying rate and the small intestine propulsion rate of the three groups were detected right after the EA intervention, and the serum expression levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured using enzyme-linked immunoassays. Real-time fluorescence quantitative polymerase chain reactions were used to detect the transcription levels of IL-1β and IL-6 in the rats′ duodenums, while western blotting was employed to assess the expression of caspase-1 P20 and dermatin D (GSDMD) in their duodenums.Results:After successful modeling, the average body weight of the rats in the model and EA groups was significantly different from that in blank group, and after 7 and 14 days the average body weight of the former groups was significantly different from that of the blank group, with significant differences between the two groups as well. After the EA intervention significant differences were observed in gastric reside and small intestine propulsion rate between the EA group and the model group, as well as between the model and the blank group. After the intervention, there were significant differences between the blank group and the other two groups in the average expression of IL-1β and IL-6 in serum, IL-1β and IL-6 mRNA in the duodenum, as well as the GSDMD and caspase-1 p20 proteins in the duodenum. There were significant differences between the model and EA groups in all of the above measurements.Conclusions:EA at the Zusanli point can significantly reduce the level of scorch death in the duodenum of FD rats, as well as relieve low-grade duodenal inflammation and the clinical symptoms of FD. Its mechanism may be related to the down-regulation of the expression of caspase-1 P20 and GSDMD-N protein, and of inflammatory factors such as IL-1β and IL-6, relieving low-grade duodenal inflammation.

Myocardial protective effect of Yangxin dingji capsule on diabetic cardiomyopathy model golden hamsters / 中国药房

OBJECTIVE To explore t he protective mechanism of Yangxin dingji capsules on the cardiomyocytes of diabetic cardiomyopathy（DCM）model golden hamsters. METHODS In this study ，golden hamsters were divided into control group （n＝ 10，no modeling ，no drug administration ），model group （n＝9，modeling，no drug administration ），TCM high-dose group [ n＝8， modeling，Yangxin dingji capsules 2 g/（kg·d）]，TCM low-dose group [ n＝8，modeling，Yangxin dingji capsules 1 g/（kg·d）] and empagliflozin group [ n＝9，positive control ，modeling，10 mg/（kg·d）]. All the golden hamsters were gavaged continuously for 8 weeks. The general conditions of golden hamsters were observed during the experiment. Blood glucose ，total cholesterol （TC）and creatine kinase MB （CK-MB），ejection fraction （EF），fractional shortening （FS），interleukin 1β（IL-1β）and transforming growth factor β1（TGF-β1）were detected ；the histopathological changes of myocardium were observed. mRNA and protein expression of nucleotide-binding oligomerization domain-like receptor protein 3（NLRP3），caspase-1，aspirin D （GSDMD），nuclear factor κB （NF-κB）and IL- 1β were detected and observed；DNA damage in myocardial was detected. RESULTS Compared with control group，the blood glucose ，TC，CK-MB，serum IL- 1β，TGF-β1 levels，the mRNA expressions and positive protein expression of NLRP 3，caspase-1，GSDMD，NF-κ B and IL-1 β and protein expression of GSDMD in golden hamsters were significantly increased in model group （P＜0.05 or P＜0.01） EF and FS were significantly decreased （P＜0.01）；the fibers of myocardial cells was disordered ， and the blue-stained collagen fibers between the myocardium increased ； DNA damaged positive cells in myocardial tiss ue of gold hamsters increased significantly. Compared with model group，the above indexes of administration groups were reversed to varying degrees ；the gap of myocardial cells were clear ，and the fibers disorder was improved ；the DNA damaged positive cells in the myocardial tissue were reduced to varying degrees. CONCLUSIONS Yangxin dingji capsule can inhibit the cardiomyocyte pyroptosis and relieve the inflammatory injury of DCM in DCM model golden hamsters by regulating the NLRP 3/caspase-1/GSDMD signaling pathway ，so as to protect the cardiomyocytes.

Block nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome to alleviate non-alcoholic steatohepatitis and liver fibrosis / 中华消化杂志

Objective:To explore the role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in non-alcoholic steatohepatitis (NASH).Methods:The liver tissue samples of 24 patients admitted the Second Affiliated Hospital of Fujian Medical University were selected, including 12 NASH samples from liver biopsy and 12 normal liver tissues from the margin of hepatic hemangioma. The expression of NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, interleukin (IL)-1β and the content of triglyceride (TG) were detected. Wild-type and NLRP3 -/- C57BL/6 mice were fed with normal diet or methionine-choline deficient diet (MCD) for 8 weeks. The wild-type mice were divided into MCC950 NASH, 0.9% sodium chloride (NaCl) NASH, MCC950 and 0.9% NaCl group, 8 mice in each group, and were fed with MCD diet and treated with MCC950, fed with MCD diet and treated with 0.9% NaCl, fed with normal diet and treated with MCC950, and fed with normal diet and treated with 0.9% NaCl respectively for eight weeks. After eight weeks, the pathologic changes of liver tissues were observed with hematoxylin-eosin staining and immunohistochemistry. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), free fatty acid (FFA), IL-1β and TG in serum were determined by enzyme linked immunosorbent assay. The expression levels of NLRP3, ASC, caspase-1 and IL-1β in liver tissues were examined by Western blotting and real-time quantitative polymerase chain reaction. Primary Kupffer cells were isolated and cultured from the livers of wild-type and NLRP3 -/- mice and divided into control group and palmitic acid group. The expression levels of related proteins in the supernatant of cells culture were detected by Western blotting. Independent sample t test and one-way ANOVA were used for statistical analysis. Results:The expression levels of NLRP3, ASC, caspase-1, IL-1β and the content of TG of the liver tissues of the NASH patients were all higher than those of healthy control group (all P<0.05). The formation of steatohepatitis in hepatocyte of MCD-fed mice was more obvious than that of nomal diet-fed mice, with more hepatocyte ballooning and inflammatory cell infiltration. The expression of NLRP3, ASC, caspase-1, IL-1β, caspase-1 activity and the content of TG in liver tissue of NASH mice were all higher than those of normal diet-fed group (all P<0.05); and serum levels of ALT, AST, IL-1β, and the content of FAA were all higher than those of normal diet-fed group (all P<0.05). The serum levels of ALT, AST, IL-1β and IL-18 of NLRP3 -/- NASH mice were all lower than those of wild-type NASH mice (all P<0.05). The serum level of ALT, the expression of ASC, caspase-1 and IL-1β in liver tissues, and the degrees of liver fibrosis of wild-type MCC950 NASH group were all lower than those of 0.9% NaCl NASH group (all P<0.05). The expression of NLRP3, ASC, caspase-1, caspase-1 activity, and secretion of IL-1β and IL-18 in Kupffer cells from wild-type mouse treated with palmitic acid were all higher than those of the negative control group (all P<0.05). However, the changes of the above indicators in Kupffer cells from NLRP3 -/- mouse were not affected by palmitic acid treatment. Conclusion:NLRP3 blockade can significantly alleviate the liver injury and fibrosis in NASH mice and prevent the development of NASH.

Effects of chitinase-3-like protein 1 on the expression of inflammatory damage-related molecules in mouse skeletal muscle satellite cells induced by lipopolysaccharide / 中华危重病急救医学

Objective:To explore the potential mechanism of chitinase-3-like protein 1 (CHI3L1) involved in skeletal muscle stem cell injury induced by sepsis.Methods:Six different concentrations of lipopolysaccharide (LPS) were used to stimulate mouse skeletal muscle satellite cells cultured in vitro. Enzyme linked immunosorbent assay (ELISA) and cell counting kit-8 (CCK-8) were used to determine the optimal concentration. The overexpression and interference vectors of CHI3L1 were constructed to transfect skeletal muscle satellite cells, and the transfection efficiency was verified by polymerase chain reaction (PCR) and Western blotting. The cells were randomly divided into blank control group (cells without any intervention), model group (LPS-stimulated untransfected cells), overexpressing CHI3L1 group (LPS-stimulated cells transfected with CHI3L1 plasmid), overexpressing CHI3L1 control group [LPS-stimulated cells transfected with negative control (NC) plasmid], CHI3L1 interference group [LPS-stimulated cells transfected with CHI3L1 small interfering RNA (siRNA)], CHI3L1 interference control group (LPS-stimulated cells transfected with CHI3L1-siRNA NC). The levels of extracellular caspase-1 and interleukin-1β (IL-1β) were detected by ELISA. The protein expressions of intracellular IL-1β, signal transducer and activator of transcription 3 (STAT3), protein kinase B (Akt) and phosphorylated Akt (p-Akt) were detected by Western blotting. Results:According to the results of CCK-8 and ELISA, the best concentration of 5 mg/L LPS was selected for the subsequent experiment. The transfection was validated by PCR and Western blotting. Compared with the blank control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular Akt, p-Akt, and IL-1β were significantly increased in the model group [IL-1β (ng/L): 11.22±0.55 vs. 8.63±0.63, caspase-1 (pmol/L): 9.47±0.22 vs. 8.65±0.15, Akt/GAPDH: 1.36±0.12 vs. 1.06±0.15, p-Akt/GAPDH: 0.78±0.07 vs. 0.09±0.01, IL-1β/GAPDH: 1.38±0.12 vs. 0.18±0.03, all P < 0.05]. Compared with the model group and the overexpressing CHI3L1 control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular p-Akt and IL-1β were significantly increased in the overexpressing CHI3L1 group [IL-1β(ng/L): 14.93±0.97 vs. 11.22±0.55, 9.38±0.40, caspase-1 (pmol/L): 10.35±0.03 vs. 9.47±0.22, 8.46±0.24, p-Akt/GAPDH: 1.21±0.04 vs. 0.78±0.07, 0.63±0.04, IL-1β/GAPDH: 1.87±0.08 vs. 1.38±0.12, 1.51±0.17, all P < 0.05]. Compared with the model group and the CHI3L1 interference control group, the levels of extracellular IL-1β, caspase-1 and the protein expressions of intracellular p-Akt and IL-1β were significantly decreased in the CHI3L1 interference group [IL-1β(ng/L): 8.98±0.73 vs. 11.22±0.55, 10.44±0.65, caspase-1 (pmol/L): 7.61±0.63 vs. 9.47±0.22, 8.37±0.38, p-Akt/GAPDH: 0.50±0.04 vs. 0.78±0.07, 0.94±0.06, IL-1β/GAPDH: 0.77±0.02 vs. 1.38±0.12, 1.13±0.07, all P < 0.05]. Conclusions:CHI3L1 may mediate the damage of skeletal muscle stem cells in sepsis by increasing the expression of caspase-1 and IL-1β. CHI3L1 may be involved in the regulation of Akt signaling pathway in skeletal muscle stem cells, but has no significant effect on STAT3 signaling pathway.