Advances in the relationship between GSDMD-mediated pyroptosis and tumor development / 中国肿瘤生物治疗杂志

@#[摘 要] 近年来研究发现gasdermin D（GSDMD）作为细胞焦亡的关键效应分子与肿瘤发生发展密切相关，GSDMD介导的细胞焦亡是一把双刃剑，一方面，焦亡引起的长期炎症反应会促进正常细胞向肿瘤细胞的转化，帮助肿瘤细胞实现免疫逃逸，促进肿瘤生长和转移；另一方面，GSDMD会介导肿瘤细胞的焦亡，引起的炎性因子释放招募免疫细胞至病变部位，同时调控肿瘤细胞增殖，抑制肿瘤的发生发展。GSDMD介导的细胞焦亡具有抗肿瘤治疗的潜在价值，目前，多种抗癌药物通过GSDMD触发细胞焦亡发挥抗肿瘤作用，同时GSDMD介导的细胞焦亡也为免疫检查点治疗提供新思路，具有广阔的研究前景。

Regulation of Pyroptosis in Gastric Cancer by Traditional Chinese Medicine： A Review / 中国实验方剂学杂志

Pyroptosis， a new type of inflammatory programmed cell death， is different from apoptosis， necrosis， cytosis， ferroptosis， and autophagy. Pyroptosis is dependent on the activation of cysteine aspartate-specific protease （Caspase）， which cleaves key mediator proteins to form pores in the cell membrane and induces the maturation and release of the proinflammatory cytokines interleukin-1β and interleukin-18 into the extracellular environment， resulting in a cascade of inflammatory reactions. Gastric cancer as a malignant tumor of the digestive tract is refractory and has poor prognosis， and the chemoradiotherapy of this disease may lead to a variety of complications. At present， the pathogenesis of gastric cancer remains unclear. Studies have proved that pyroptosis is associated with the occurrence and development of gastric cancer， which has attracted wide attention. Pyroptosis is a double-edged sword for gastric cancer. On the one hand， it can release the contents of proinflammatory cells to amplify or maintain inflammation and induce the "inflammation-cancer" transformation of cells. On the other hand， pyroptosis can enhance the sensitivity of drugs for chemotherapy to improve the therapeutic effect and survival. In recent years， the anti-tumor mechanism of traditional Chinese medicine （TCM） has become a research hotspot as TCM has demonstrated significant effects in clinical application. Therefore， the regulation of pyroptosis by TCM may be a new direction for the treatment of gastric cancer in the future. Based on the available studies， this paper introduces the roles of pyroptosis-associated key proteins in the occurrence and development of gastric cancer. Furthermore， this paper summarizes the effects of TCM prescriptions and active ingredients on alleviating gastric mucosal damage， reducing the incidence of gastric cancer， and preventing tumor metastasis and recurrence by mediating pyroptosis pathways， aiming to provide new ideas for deciphering the mechanism of pyroptosis and exploring the TCM treatment of gastric cancer in the future.

Natural Medicinal Components Mediating Pyroptosis by GSDMs in Anti-tumor Therapy： A Review / 中国实验方剂学杂志

Pyroptosis， an atypical new cell death mode other than apoptosis and necrosis， has been discovered in recent years. Pyroptosis depends on the cleavage of gasdermins （GSDMs） by Caspases. The activated GSDMs act on the plasma membrane to form a perforation， which results in cell lysis and triggers inflammation and immune response. Pyroptosis can be induced by four distinct signaling pathways， including canonical and non-canonical inflammasome pathways， apoptosis-associated Caspases-mediated pathway， and granzyme pathway. In these signaling pathways， GSDMs are the executors of pyroptosis. Pyroptosis is associated with the death of tumor cells and the inflammatory damage of normal tissues. Recent studies have demonstrated that moderate pyroptosis can lead to tumor cell death to exert an anti-tumor effect， and meanwhile stimulate the tumor immune microenvironment， while it can promote tumor development. Despite the good performance， drug-based anti-tumor therapies such as tumor immunotherapy， chemotherapy， and targeted therapy have some shortcomings such as drug resistance， recurrence， and damage to normal tissues. The latest research shows that a variety of natural compounds have anti-tumor effects in the auxiliary treatment of tumors by mediating the pyroptosis pathways in a multi-target and multi-pathway manner， which provide new ideas for the study of anti-tumor therapy. We reviewed the molecular mechanism of pyroptosis and the regulatory role of pyroptosis in tumors and tumor immune microenvironment， and summarized the recent research progress in the natural medicinal components regulating pyroptosis in anti-tumor therapy， with a view to providing ideas for the research on the anti-tumor therapy based on pyroptosis.

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.

GSDMD in peripheral myeloid cells regulates microglial immune training and neuroinflammation in Parkinson's disease

Peripheral bacterial infections without impaired blood-brain barrier integrity have been attributed to the pathogenesis of Parkinson's disease (PD). Peripheral infection promotes innate immune training in microglia and exacerbates neuroinflammation. However, how changes in the peripheral environment mediate microglial training and exacerbation of infection-related PD is unknown. In this study, we demonstrate that GSDMD activation was enhanced in the spleen but not in the CNS of mice primed with low-dose LPS. GSDMD in peripheral myeloid cells promoted microglial immune training, thus exacerbating neuroinflammation and neurodegeneration during PD in an IL-1R-dependent manner. Furthermore, pharmacological inhibition of GSDMD alleviated the symptoms of PD in experimental PD models. Collectively, these findings demonstrate that GSDMD-induced pyroptosis in myeloid cells initiates neuroinflammation by regulating microglial training during infection-related PD. Based on these findings, GSDMD may serve as a therapeutic target for patients with PD.

Jinyinqingre Oral Liquid alleviates LPS-induced acute lung injury by inhibiting the NF-κB/NLRP3/GSDMD pathway / 中国天然药物

Acute lung injury (ALI) is a prevalent and severe clinical condition characterized by inflammatory damage to the lung endothelial and epithelial barriers, resulting in high incidence and mortality rates. Currently, there is a lack of safe and effective drugs for the treatment of ALI. In a previous clinical study, we observed that Jinyinqingre oral liquid (JYQR), a Traditional Chinese Medicine formulation prepared by the Taihe Hospital, Affiliated Hospital of Hubei University of Medicine, exhibited notable efficacy in treating inflammation-related hepatitis and cholecystitis in clinical settings. However, the potential role of JYQR in ALI/acute respiratory distress syndrome (ARDS) and its anti-inflammatory mechanism remains unexplored. Thus, the present study aimed to investigate the therapeutic effects and underlying molecular mechanisms of JYQR in ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI and an in vitro RAW264.7 cell model. JYQR yielded substantial improvements in LPS-induced histological alterations in lung tissues. Additionally, JYQR administration led to a noteworthy reduction in total protein levels within the BALF, a decrease in MPAP, and attenuation of pleural thickness. These findings collectively highlight the remarkable efficacy of JYQR in mitigating the deleterious effects of LPS-induced ALI. Mechanistic investigations revealed that JYQR pretreatment significantly inhibited NF-κB activation and downregulated the expressions of the downstream proteins, namely NLRP3 and GSDMD, as well as proinflammatory cytokine levels in mice and RAW2647 cells. Consequently, JYQR alleviated LPS-induced ALI by inhibiting the NF-κB/NLRP3/GSDMD pathway. JYQR exerts a protective effect against LPS-induced ALI in mice, and its mechanism of action involves the downregulation of the NF-κB/NLRP3/GSDMD inflammatory pathway.

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.

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.

A preliminary study on Candida albicans-induced pyroptosis of murine bone marrow-derived macrophages / 中华皮肤科杂志

Objective:To investigate the effect of Candida albicans ( C. albicans) on pyroptosis of murine bone marrow-derived macrophages (BMDMs) . Methods:Live-cell imaging was used to observe morphologic changes of in vitro C. albicans-infected BMDMs (multiplicity of infection [MOI] = 50) so as to evaluate whether pyroptosis occurred. Cultured BMDMs were divided into a control group and a C. albicans group, which were treated with phosphate-buffered saline and C. albicans suspensions respectively for 6 hours; then, real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of NOD-like receptor pyrin domain containing 3 (NLRP3), interleukin (IL) -1β and IL-18, and Western blot analysis to determine the protein expression and cleavage levels of NLRP3, caspase-1 and gasdermin D (GSDMD). BMDMs were cultured with C. albicans suspensions for different durations (0, 10, 15, 20, and 25 hours), and enzyme-linked immunosorbent assay was conducted to detect secretion levels of IL-1β and IL-18. Cultured wild-type BMDMs and GSDMD-knockout BMDMs were treated with C. albicans suspensions for 15 minutes, and then rates of phagocytosis of C. albicans by wild-type BMDMs and GSDMD-knockout BMDMs were estimated by flow cytometry; after 6-hour treatment with C. albicans, flow cytometry and lactate dehydrogenase (LDH) release assay were performed to assess mortality rates of wild-type BMDMs and GSDMD-knockout BMDMs. In addition, some wild-type BMDMs and GSDMD-knockout BMDMs were separately divided into blank control group, control group, maximum enzyme activity-sample control group, IL-1β alone group, C. albicans alone group, and IL-1β + C. albicans group, and cell mortality rates were detected by the LDH release assay after treatment with IL-1β and/or C. albicans. Statistical analysis was carried out by using unpaired t test, Kruskal-Wallis test, analysis of variance, and other statistical methods. Results:After in vitro treatment with C. albicans, swelling and ballooning with large bubbles blowing from the plasma membrane occurred in BMDMs, suggesting the occurrence of cell pyroptosis; compared with the control group, the C. albicans group showed significantly increased mRNA expression levels of NLRP3 and IL-1β after 6-hour treatment with C. albicans ( t = 13.02, 17.51, respectively, P = or < 0.001), but no significant change in the IL-18 mRNA expression level ( P = 0.486), and Western blot analysis showed that C. albicans could increase the expression of NLRP3 inflammasomes, as well as cleaved caspase-1 and GSDMD. After the treatment with C. albicans for different durations (0, 10, 15, 20, and 25 hours), the secretion level of IL-1β by BMDMs gradually increased over time ( H = 12.90, P = 0.012), while the secretion level of IL-18 did not significantly change ( F = 0.48, P = 0.753), and the secretion level of IL-1β was significantly lower in the GSDMD-knockout BMDM group than in the wild-type BMDM group ( F = 24.22, P = 0.008). After 15-minute in vitro treatment with C. albicans, the phagocytosis rate of C. albicans was significantly lower in the GSDMD-knockout BMDM group (50.3% ± 1.10%) than in the wild-type BMDM group (58.53% ± 1.19%, t = 5.09, P = 0.007) ; after 6-hour treatment with C. albicans, the cell mortality rate was significantly higher in the GSDMD-knockout BMDM group than in the wild-type BMDM group (flow cytometry: 38.40% ± 0.50% vs. 34.37% ± 0.52%, t = 4.72, P = 0.009; LDH release assay: 22.52% ± 0.18% vs. 12.48% ± 0.15%, t = 42.36, P < 0.001) ; the cell mortality rates of wild-type BMDMs and GSDMD-knockout BMDMs both significantly decreased in the IL-1β + C. albicans groups compared with the C. albicans groups (both P < 0.001) . Conclusion:Pyroptosis could be induced in murine BMDMs after C. albicans infection, which promotes the release of IL-1β and may reduce the mortality rate of macrophages by improving their immune activity.

JNK inhibitor SP600125 inhibits interleukin-1β induced pyroptosis in βTC-6 cells / 中华内分泌代谢杂志

Objective:To investigate whether interleukin(IL)-1β is involved in pyroptosis which leads to mouse islet β cell line βTC-6 cell damage, and to explore the role of JNK inhibitor SP600125 in inhibiting IL-1β induced βTC-6 cell pyroptosis.Methods:βTC-6 cell line and mouse islets were incubated with IL-1β for 48 h or intervened with both JNK inhibitor SP600125 and IL-1R antagonist IL-1Ra, then GSDMD expression and β cell pyroptosis morphology were detected by immunofluorescence staining of GSDMD and DAPI. The expression levels of Gsdmd, IL-1β and IL-18 mRNAs were detected by real time fluorescence PCR, and apoptosis was examined by Annexin-V/7-AAD staining combined with flow cytometry.Results:βTC-6 cell pyroptotic body was significantly increased in the IL-1β treated group compared with the control group, and the expressions of pyroptosis related genes Gsdmd, IL-1β, and IL-18 mRNA were significantly higher( P<0.05), and apoptosis was increased, suggesting that IL-1β effectively induced the βTC-6 cell pyroptosis, IL-1Ra prevented IL-1β induced βTC-6 cell pyroptosis. In the presence of JNK inhibitor SP600125, IL-1β treatment failed to induce the expressions of Gsdmd and IL-18 mRNA, markers of pyroptosis, and reduced the rate of apoptosis, indicating that SP600125 suppressed IL-1β induced βTC-6 cell pyroptosis. Conclusion:Pyroptosis is one of the mechanisms of βTC-6 cell impairment caused by IL-1β, and SP600125, a JNK inhibitor, can block the IL-1β induced pyroptosis pathway and has a potential role in inhibiting βTC-6 cell pyroptosis.

Effect of HMGB1/Caspase-1/GSDMD signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury / 器官移植

Objective To evaluate the effect of high mobility group box 1 (HMGB1)/ cysteinyl aspartate specific proteinase (Caspase)-1/Gasdermin D (GSDMD) signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury (IRI). Methods C57BL/6 mice were randomly divided into the sham operation group (Sham group), IRI 2 h group, IRI 6 h group, IRI 12 h group, glycyrrhizic acid (GA)+Sham group and GA+IRI 12 h group (n=8 in each group). AML12 cells were evenly divided into the Sham group, IRI 12 h group, GA+Sham group and GA+IRI 12 h group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1β and IL-6 in each group were detected by enzyme-linked immune absorbent assay(ELISA). The messenger ribonucleic acid (mRNA) levels of IL-1β and IL-6 were detected by reverse transcription polymerase chain reaction(RT-PCR). The pathological score of liver ischemia and cell apoptosis were compared among all groups. The expression level of HMGB1 in the liver tissues of each group was determined by immunohistochemistry. The expression levels of HMGB1, Caspase-1 and GSDMD proteins in the mouse liver tissues and AML12 cells were measured by Western blot. Results Compared with the Sham group, the serum levels of ALT, AST, IL-1β and IL-6 and the relative expression levels of IL-1β and IL-6 mRNA in the liver tissues were all significantly up-regulated after IRI in each group (all P < 0.05), and showed significant time-dependent pattern along with the prolongation of reperfusion time. Compared with the Sham group, the pathological score of hepatic ischemia and the apoptosis rate of hepatocytes were significantly increased after IRI in each group (all P < 0.05). Immunohistochemical results showed that the expression level of HMGB1 in the liver tissues was significantly up-regulated after IRI, which showed an increasing trend along with the prolongation within the period of 2-12 h. Western blot showed that compared with the Sham group, the relative expression levels of HMGB1, Caspase-1 and GSDMD proteins in vivo and in vitro were up-regulated in the IRI 12 h group. The relative expression level of HMGB1 protein was significantly up-regulated, whereas those of Caspase-1 and GSDMD proteins were significantly down-regulated in the GA+IRI 12 h group compared with those in the IRI 12 h group (all P < 0.05). Conclusions Hepatocytes probably activate the Caspase-1/GSDMD signaling pathway by releasing HMGB1, thereby triggering hepatocyte pyroptosis and leading to liver IRI. Inhibition of extracellular release of HMGB1 by GA may mitigate liver IRI.

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.

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.

Research progress on induction of pyroptosis in antitumor therapy / 中国肿瘤生物治疗杂志

@#[摘 要] 细胞焦亡是近年来发现的一种新型细胞死亡的方式，是一种受焦孔素（GSDM）家族调控的炎症性程序性细胞死亡，其主要特征是膜穿孔、细胞肿胀及细胞破裂。细胞焦亡发生的机制分为由GSDMD介导的Caspase-1和Caspase-4/-5/-11依赖性经典炎症小体途径和由GSDME介导的Caspase-3和颗粒酶依赖性非经典炎症小体途径等。近年来研究显示，细胞焦亡具有抑制和促进肿瘤发生发展的双重作用，并且细胞焦亡的诱导在抗肿瘤免疫治疗中也发挥双重效应：一方面通过促进炎症因子释放，形成肿瘤微环境，抑制抗肿瘤免疫，另一方面则通过引发抗肿瘤炎症反应抑制肿瘤细胞的增殖。此外，细胞焦亡的诱导在化疗及其他联合治疗中也发挥着重要作用。进一步研究发现，中药及其提取物调控细胞焦亡的诱导对于治疗肿瘤至关重要。

Expression and significance of non-canonical pyroptosis pathway-related proteins in muscle tissues of patients with dermatomyositis/polymyositis / 中华微生物学和免疫学杂志

Objective:To detect the expression of cysteinyl aspartate specific proteinase 4 (caspase-4), caspase-5, gasdermin D (GSDMD), nucleotide-binding oligomerization domain-like receptor protein-3 (NLRP3), Pannexin-1 and P2X7 involved in non-canonical pyroptosis pathway in muscle tissues of patients with dermatomyositis (DM)/polymyositis (PM) and to investigate the roles and significance of them in the pathogenesis of DM and PM.Methods:Altogether 13 DM patients, nine PM patients and 20 volunteers (control group) treated in the Affiliated Hospital of Qinghai University from January 2019 to September 2020 were enrolled in the present study. The 20 volunteers with no additional concomitant diseases underwent debridement due to simple orthopedic trauma. Pathological changes in muscle tissues were detected by hematoxylin & eosin (HE) staining. Expression of caspase-4, caspase-5, GSDMD, NLRP3, Pannexin-1 and P2X7 in muscle tissues was measured using immunohistochemistry (IHC).Results:(1) HE staining results showed that the muscle fibers in the control group had basically normal morphology and structure with no obvious inflammatory cell infiltration, atrophy, degeneration or necrosis. However, the size and thickness of muscle fibers in DM and PM groups were different with excessive inflammatory cell infiltration, atrophy, degeneration and necrosis to varying degrees. Moreover, the pathological scores of HE staining in muscle tissues of DM and PM groups were significantly higher than that of the control group and the differences were of statistical significance ( P<0.05). (2) IHC staining results suggested that the expression of caspase-4, caspase-5, GSDMD, NLRP3, Pannexin-1 and P2X7 in muscle tissues was higher in DM and PM groups than in the control group ( P<0.05). (3) As indicated by Pearson correlation analysis, the pathological scores of HE staining in muscle tissues of DM and PM groups were positively correlated with the IHC scores of caspase-4, caspase-5, GSDMD, NLRP3, PAnnexin-1 and P2X7 ( P<0.05). Furthermore, the IHC scores of caspase-4 and caspase-5 were positively correlated with the IHC scores of GSDMD and Pannexin-1 ( P<0.05); the IHC score of GSDMD was positively correlated with the IHC score of NLRP3 ( P<0.05); the IHC score of Pannexin-1 was positively correlated with the IHC score of P2X7 in muscle tissues ( P<0.05). Conclusions:The non-canonical pyroptosis pathway might be involved in the pathogenesis of DM and PM, which was possibly achieved by promoting inflammatory response. These results suggested that the non-canonical pyroptosis pathway played crucial roles in the immune pathogenesis of DM and PM.

Protective effect of GSDMD gene knock-out on acute lung injury induced by LPS in mice / 解放军医学杂志

Objective: To investigate the potential function of GSDMD in lipopolysaccharide (LPS) induced acute lung injury (ALI) during the inflammatory reaction and lung tissue damage. Methods: Twenty wild-type C57BL/6 mice and twenty GSDMD knockout C57BL/6 mice were randomized into the WT-sham group, WT-ALI group, Knockout (KO)-sham group and KOALI group. The ALI was induced by intratracheal injection of LPS (1 mg/kg), using PBS as the treatment control. To examine the inflammatory reaction and tissue damage in the lung, the lung tissue and bronchoalveolar lavage fluid (BALF) were collected. HE staining of lung tissue and the semi-quantitative score of lung injury were performed. The wet/dry ratio of lung tissue and the total protein concentration in BALF were measured by BCA. The levels of TNF-a, IL-1β, and IL-6 in BALF were detected by ELISA. The expression of pyroptosis-related proteins in BALF and lung tissues were detected by Western blotting. Results: Compared with the WT-ALI group, the lung injury pathological score, wet/dry ratio, and total protein concentration were all significantly lower in KO-ALI group (P<0.01). Furthermore, the total protein level, TNF-a, IL-1β, and IL-6 levels in BALF were significantly lower in KO-ALI group (P<0.05 or P<0.01). There was no GSDMD and GSDMD-NT expression in the KO mice. After LPS stimulation, the expression levels of caspase-1 and caspase-11 were up-regulated in both wild-type and knockout mice (P<0.01). Conclusions: The expression and activation of GSDMD protein in the lung tissue of mice could be induced by intratracheal instillation of LPS and knockout of GSDMD can inhibit the inflammation level of the lung tissue and reduce its damage degree in mice.

Establishment and significance of pyrolysis model of human trophoblast HTR-8/SVneo induced by hydrogen peroxide / 医学研究生学报

ObjectiveA good invasion ability of extravilloustrophoblas (EVTs) is the prerequisite for successful placental colonization and effective remodeling of the uterine spiral artery. This article aims to simulate the pathophysiological process of oxidative stress inducing trophoblasts to pyroptosis in vitro, exploring the correlation between trophoblasts pyroptosis and the pathogenesis of preeclampsia.MethodsTwenty-five patients with preeclampsia were selected from the Department of Obstetrics and Gynecology, Zhongda Hospital affiliated to Southeast University from September 2017 to January 2019. Among them, early-onset preeclampsia (gestational weeks<34) was early-onset group (n=17), late-onset preeclampsia (gestational weeks≥34) was late-onset group (n=8), and full-term pregnant women with normal blood pressure (39＜gestational weeks＞42) were selected as normal group (n=10). Human trophoblasts were cultured with HTR-8/SVneo for 12 hours, and then treated with H2O2 (100, 150, 200, 250μmol/L) (2, 4, 6, 12 h), to induce human trophoblast HTR-8/SVneo pyrolysis model; the control group was normal cultured cells of 1640+10% fetal bovine serum + 1% antibiotics. Placental specimens from 7 patients with preeclampsia were randomly selected, including 3 cases in early onset group, 4 cases in late onset group and 1 case in normal group. The total proteins of cells and placenta were extracted respectively, and the expression of scorch death-related molecular proteins was detected. The mRNA levels of pyroptosis related molecules in cells was detected by RT-qPCR, and the morphological changes of cells were observed by inverted phase contrast microscope.ResultsThe Western blot results showed that the activation of the key molecular activation form of the cell pyrogenesis pathway, Cleaved caspase1, could be detected in the placenta. When H2O2 was 150 mol/L for 2h, the mRNA levels of NLRP3 and IL-1, the key molecules of the upstream activation signal, were significantly up-regulated (8.680±0.481, 14.136±0.244) compared with the control group (1.00±0.00) (P<0.000). At 4h, mRNA levels of key molecule GSDMD and downstream inflammatory factor IL-18 (1.639±0.354 and 1.794±0.043) in the pyrogenesis pathway were significantly higher than those in the control group (1.00±0.00), with statistically significant differences (P<0.05). By reverse validation of the mRNA levels of the molecules associated with pyroptosis, the optimal conditions of the model induced by H2O2 were 150 mol/L and 4h, and the typical changes, such as cell swelling, fragmentation and plasma membrane bubble formation, could be seen under the light microscope.ConclusionThe pyroptosis model of trophoblast cells was successfully established, and the physiological process of oxidative stress inducing trophoblasts to pyroptosis in vitro was successfully simulated, providing new ideas and directions for the diagnosis and treatment of preeclampsia and the development of new drugs.

Research progress in Gasdermin family proteins and their association with diseases / 中华微生物学和免疫学杂志

Gasdermin family (GSDMs), consisting of six proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) in humans and ten proteins (GSDMA1-3, GSDMC1-4, GSDMD, GSDME and DFNB59) in mice, might be involved in multiple physiological and pathological processes, including epithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. Recent studies confirmed GSDMD, which containing an N-terminal domain with pore-forming activity and a C-terminal domain with structural autoinhibition, as a crucial substrate of inflammatory caspases in pyroptosis, pioneering a new area for structural and functional research on Gasdermin family proteins. This review will summarize the latest progress in the structures, functions and association with diseases of several Gasdermin family proteins.

Research progress in Gasdermin family proteins and their association with diseases / 中华微生物学和免疫学杂志

Gasdermin family (GSDMs), consisting of six proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and DFNB59) in humans and ten proteins (GSDMA1-3, GSDMC1-4, GSDMD, GSDME and DFNB59) in mice, might be involved in multiple physiological and pathological processes, including ep-ithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. Recent studies confirmed GSDMD, which containing an N-terminal domain with pore-forming activity and a C-termi-nal domain with structural autoinhibition, as a crucial substrate of inflammatory caspases in pyroptosis, pio-neering a new area for structural and functional research on Gasdermin family proteins. This review will sum-marize the latest progress in the structures, functions and association with diseases of several Gasdermin fam-ily proteins.

Gasdermin D:The killer protein performs cell pyroptosis / 医学研究生学报

Pyroptosis is a novel programmed cell death mode mediated by inflammatory Caspase,which has a proinflammatory effect and is accompanied by cell membrane rupture and disintegration.In recent years,researchers found that the substrate of inflam-matory Caspase-Gasdermin D(GSDMD)protein,is the key"killer protein"to execute cell pyroptosis.The inflammatory Caspase spe-cifically cleaves the GSDMD protein,forming the N-terminal domain and the C-terminal domain,and eventually results in cell pyropto-sis by protein pores formation.This article focuses on the research progress of cell pyroptosis,the structural features of GSDMD protein, and the mechanism of GSDMD protein participating in the pyroptosis and the disease models.