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.

Effect of Polygonati Rhizoma in improving pyroptosis injury of diabetic macroangiopathy via NLRP3/caspase-1/GSDMD pathway / 中国中药杂志

This study aims to explore the influence of Polygonati Rhizoma on the pyroptosis in the rat model of diabetic macroangiopathy via the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/gasdermin D(GSDMD) pathway. The rat model of diabetes was established by intraperitoneal injection of streptozotocin(STZ) combined with a high-fat, high-sugar diet. The blood glucose meter, fully automated biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, and Western blot were employed to measure blood glucose levels, lipid levels, vascular thickness, inflammatory cytokine levels, and expression levels of pyroptosis-related proteins. The mechanism of pharmacological interventions against the injury in the context of diabetes was thus explored. The results demonstrated the successful establishment of the model of diabetes. Compared with the control group, the model group showed elevated levels of fasting blood glucose, total cholesterol(TC), triglycerides(TG) and low-density lipoprotein cholesterol(LDL-c), lowered level of high-density lipoprotein cholesterol(HDL-c), thickened vascular intima, and elevated serum and aorta levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-18(IL-18). Moreover, the model group showed increased NLRP3 inflammasomes and up-regulated levels of caspase-1 and GSDMD in aortic vascular cells. Polygonati Rhizoma intervention reduced blood glucose and lipid levels, inhibited vascular thickening, lowered the levels of TNF-α, IL-1β, IL-18 in the serum and aorta, attenuated NLRP3 inflammasome expression, and down-regulated the expression levels of caspase-1 and GSDMD, compared with the model group. In summary, Polygonati Rhizoma can slow down the progression of diabetic macroangiopathy by inhibiting pyroptosis and alleviating local vascular inflammation.

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.

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.

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.

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.

Effect of Huangqin Decoction on pyroptosis pathway of NLRP3/caspase-1 in mice with ulcerative colitis / 中国中药杂志

To explore the effect of Huangqin Decoction on ulcerative colitis(UC) pyroptosis, and to explain the mechanism of pyroptosis based on NOD-like receptor thermoprotein domain 3(NLRP3)/cysteine proteinase 1(caspase-1) pathway. The animal model of UC induced with 3% dextran sodium sulfate(DSS) was established. The experimental animals were divided into control group, model group, low-dose(4.55 g·kg~(-1)), medium-dose(9.1 g·kg~(-1)) and high-dose(18.2 g·kg~(-1)) Huangqin Decoction groups and salazosulfapyridine group(0.45 g·kg~(-1)). While modeling, intragastric administration was given for 7 consecutive days. On the 8 th day, the mice were euthanized, the colon length was collected, and the histopathological changes were observed by HE staining. The content of interleukin-18(IL-18) was observed by ELISA. The content of lactatedehydrogenase(LDH)was determined by microplate method. TUNEL assay kit was used to detect the cell death. The immunohistochemical staining was used to detect the expressions of NLRP3 and apoptosis-associated speck-like protein containing a CARD(ASC). Western blot was used to detect the expressions of interleukin-1β(IL-1β), caspase-1 and gasdermin D(GSDMD).The experimental study showed that compared with normal group, the LDH content, TUNEL positive staining, inflammatory factors(IL-18, IL-1β), and proteins associated with pyroptosis were significantly increased(P<0.05). Compared with model control group, the LDH content, TUNEL positive staining, inflammatory factors(IL-18, IL-1β), and proteins associated with pyroptosis were decreased, and these results were more significant in high-dose groups(P<0.05). The results of HE staining showed that Huangqin Decoction could improve the pathological changes of colon. Huangqin Decoction could inhibit UC cell pyroptosis, and the mechanism may be closely related to NLRP3/caspase-1 signaling pathway.

Effect of Iridoid-rich Fraction from Valeriana Jatamansi Jones on Neuron Pyroptosis in Rats with Acute Spinal Cord Injury / 中国康复理论与实践

Objective:To investigate the effects of iridoid-rich fraction from Valeriana jatamansi Jones (IRFV) on neuronal pyroptosis in rats with acute spinal cord injury, and to explain the related mechanism of neuroprotection. Methods:Twenty-four healthy male Sprague-Dawley rats were randomly divided into sham-operated group, model group and treatment group, with eight rats in each group. The model of spinal cord injury was established by using a medical aneurysm clip in the latter two groups. Only the lamina was removed without injury to the spinal cord in the sham-operated group. Four hours after the operation, the treatment group was given IRFV solution 10 mg/kg, the model group and the sham-operated group were given the same volume of sodium carboxymethyl cellulose (CMC-Na) solution, for seven days. The rats were sacrificed to detected the pathological changes and the residual area of spinal cord tissue through HE staining. The apoptosis of nerve cells of the spinal cord tissue at the perilesional area was detected by TUNEL fluorescent staining. The levels of interleukin (IL)-1 and IL-18 in serum were detected by ELISA Kit and the expression of NLRP3, Caspase-1 and GSDMD were detected by Western blotting. Results:Compared with the sham-operated group, the residual area of spinal cord tissue decreased (P < 0.05), and the positive rate of TUNEL staining, the level of IL-1 and IL-18, and the expression of pyroptosis-associated proteins (NLRP3, Caspase-1 and GSDMD) increased (P < 0.05) in the model group. Compared with the model group, the pathological condition of the spinal cord tissue improved and the residual area of the spinal cord tissue increased (P < 0.05); the positive rate of TUNEL staining, the level of IL-1 and IL-18 and the expression of NLRP3, Caspase-1 and GSDMD decreased (P < 0.05) in the treatment group. Conclusion:IRFV could attenuate the inflammatory response to exert neuroprotective effects, which may be related to the regulation of NLRP3/Caspase-1 signaling pathway to inhibit the neuronal pyroptosis in rats with acute spinal cord injury.