Objective: This study aims to investigate the associations between genetic variations of pyroptosis pathway related key genes and adverse events (AEs) of postoperative chemoradiotherapy (CRT) in patients with rectal cancer. Methods: DNA was extracted from the peripheral blood which was collected from 347 patients before CRT. Sequenom MassARRAY was used to detect the genotypes of 43 haplotype-tagging single nucleotide polymorphisms (htSNPs) in eight pyroptosis genes, including absent in melanoma 2 (AIM2), caspase-1 (CASP1), caspase-4(CASP4), caspase-5 (CASP5), caspase-11 (CASP11), gasdermin D (GSDMD), gasdermin E (GSDME) and NLR family pyrin domain containing 3 (NLRP3). The associations between 43 htSNPs and AEs were evaluated by the odd ratios (ORs) and 95% confidence intervals (CIs) by unconditional logistic regression models, adjusted for sex, age, clinical stage, tumor grade, Karnofsky performance status (KPS), surgical procedure, and tumor location. Results: Among the 347 patients with rectal cancer underwent concurrent CRT with capecitabine after surgery, a total of 101(29.1%) occurred grade ≥ 2 leukopenia. rs11226565 (OR=0.41, 95% CI: 0.21-0.79, P=0.008), rs579408(OR=1.54, 95% CI: 1.03-2.29, P=0.034) and rs543923 (OR=0.63, 95% CI: 0.41-0.98, P=0.040) were significantly associated with the occurrence of grade ≥ 2 leukopenia. One hundred and fifty-six (45.0%) had grade ≥ 2 diarrhea, two SNPs were significantly associated with the occurrence of grade ≥ diarrhea, including CASP11 rs10880868 (OR=0.55, 95% CI: 0.33-0.91, P=0.020) and GSDME rs2954558 (OR=1.52, 95% CI: 1.01-2.31, P=0.050). In addition, sixty-six cases (19.0%) developed grade ≥2 dermatitis, three SNPs that significantly associated with the risk of grade ≥2 dermatitis included GSDME rs2237314 (OR=0.36, 95% CI: 0.16-0.83, P=0.017), GSDME rs12540919 (OR=0.52, 95% CI: 0.27-0.99, P=0.045) and NLRP3 rs3806268 (OR=1.51, 95% CI: 1.03-2.22, P=0.037). There was no significant difference in the association between other genetic variations and AEs of rectal cancer patients (all P>0.05). Surgical procedure and tumor location had great impacts on the occurrence of grade ≥2 diarrhea and dermatitis (all P<0.01). Conclusion: The genetic variants of CASP4, CASP11, GSDME and NLRP3 are associated with the occurrence of AEs in patients with rectal cancer who received postoperative CRT, suggesting they may be potential genetic markers in predicting the grade of AEs to achieve individualized treatment of rectal cancer.
Humans ; Pyroptosis ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Gasdermins ; Chemoradiotherapy/adverse effects* ; Rectal Neoplasms/surgery* ; Caspases/metabolism* ; Diarrhea/chemically induced* ; Leukopenia/genetics* ; Genetic Variation ; Dermatitis

Based on network pharmacology, molecular docking, and in vitro experimental verification, this study aims to explore the effect of Albiziae Cortex-Tribuli Fructus combination on HSC-LX2 pyroptosis. Specifically, the targets of Albiziae Cortex, Tribuli Fructus, and hepatic fibrosis were retrieved from an online database and CNKI, and "drug-component-target" network and "drug-component-target-disease" network were constructed. Protein-protein interaction(PPI) network was established based on STRING. Metascape was employed for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and the mechanism of Albiziae Cortex-Tribuli Fructus combination against liver fibrosis was predicted. Molecular docking was used to verify some of the results of network pharmacology, and in vitro experiment was carried out to further verify the above conclusions. According to the results of network pharmacological analysis, 25 active components and 439 targets of Albiziae Cortex-Tribuli Fructus combination and 152 anti-liver fibrosis targets were screened out, including nucleotide-binding oligomerization domain and leucine-rich-repeat-and pyrin-domain-containing 3(NLRP3) and caspase-1. The key targets were involved in 194 KEGG pathways in which the NOD-like receptor signaling pathway topped. The binding common targets were related to pyroptosis. The results of in vitro experiment showed that the pair-containing serum reduced the proliferation rate of HSC-LX2 and the content of reactive oxygen species(ROS), interleukin-18(IL-18), and interleukin-1β(IL-1β)(P<0.05). Western blot and qRT-PCR suggested that the protein and gene expression of NLRP3, caspase-1, α-smooth muscle actin(α-SMA), and gasdermin D(GSDMD) in HSC-LX2 increased after AngⅡ stimulation, and the expression decreased after the intervention of pair-containing serum(P<0.05). In summary, the pair-containing serum can inhibit the classic pathway of pyroptosis, which may be the anti-liver fibrosis mechanism. This is consistent with the predicted results of network pharmacology.
Humans ; Hepatic Stellate Cells ; Network Pharmacology ; Molecular Docking Simulation ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/genetics* ; Fibrosis ; Drugs, Chinese Herbal/pharmacology*

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.
Animals ; Mice ; Caspase 1/genetics* ; Colitis, Ulcerative/genetics* ; Colon ; Dextran Sulfate/adverse effects* ; Disease Models, Animal ; Interleukin-10/genetics* ; Interleukin-6/genetics* ; Mesalamine/pharmacology* ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Scutellaria baicalensis/chemistry* ; Tumor Necrosis Factor-alpha/metabolism* ; Drugs, Chinese Herbal/pharmacology*

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1β, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1β, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1β, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.
Female ; Animals ; Humans ; Mice ; Candidiasis, Vulvovaginal/drug therapy* ; Inflammasomes/genetics* ; Interleukin-18 ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; 1-Butanol/pharmacology* ; Fluconazole/therapeutic use* ; Interleukin 1 Receptor Antagonist Protein/therapeutic use* ; Mice, Inbred C57BL ; Candida albicans ; Cytokines ; Drugs, Chinese Herbal/pharmacology* ; Ethanol ; RNA, Messenger ; Calcium-Binding Proteins/therapeutic use*

Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1β and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.
Humans ; Inflammasomes/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Podocytes/metabolism* ; Hepatitis B virus/genetics* ; Caspase 1/metabolism* ; Cytokines/metabolism* ; Carrier Proteins/metabolism* ; MicroRNAs/genetics* ; Glomerulonephritis/metabolism* ; RNA, Small Interfering

10,11-Dehydrocurvularin (DCV) is a natural-product macrolide that has been shown to exert anti-inflammatory activity. However, the underlying mechanism of its anti-inflammatory activity remains poorly understood. Aberrant activation of the NLRP3 inflammasome is involved in diverse inflammation-related diseases, which should be controlled. The results showed that DCV specifically inhibited the activation of the NLRP3 inflammasome in association with reduced IL-1β secretion and caspase-1 activation, without effect on the NLRC4 and AIM2 inflammasomes. Furthermore, DCV disturbed the interaction between NEK7 and NLRP3, resulting in the inhibition of NLRP3 inflammasome activation. The C=C double bond of DCV was required for the NLRP3 inflammasome inhibition induced by DCV. Importantly, DCV ameliorated inflammation in vivo through inhibiting the NLRP3 inflammasome. Taken together, our study reveals a novel mechanism by which DCV suppresses inflammation, which indicates the potential role of DCV in NLRP3 inflammasome-driven inflammatory disorders.
Animals ; Mice ; Inflammasomes ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Inflammation/drug therapy* ; Anti-Inflammatory Agents/pharmacology* ; Interleukin-1beta/genetics* ; Mice, Inbred C57BL

Gut microbiota dysbiosis is an avenue for the promotion of atherosclerosis (AS) and this effect is mediated partly via the circulating microbial metabolites. More microbial metabolites related to AS vascular inflammation, and the mechanisms involved need to be clarified urgently. Paeonol (Pae) is an active compound isolated from Paeonia suffruticoas Andr. with anti-AS inflammation effect. However, considering the low oral bioavailability of Pae, it is worth exploring the mechanism by which Pae reduces the harmful metabolites of the gut microbiota to alleviate AS. In this study, ApoE^-/- mice were fed a high-fat diet (HFD) to establish an AS model. AS mice were administrated with Pae (200 or 400 mg·kg^-1) by oral gavage and fecal microbiota transplantation (FMT) was conducted. 16S rDNA sequencing was performed to investigate the composition of the gut microbiota, while metabolomics analysis was used to identify the metabolites in serum and cecal contents. The results indicated that Pae significantly improved AS by regulating gut microbiota composition and microbiota metabolic profile in AS mice. We also identified α-hydroxyisobutyric acid (HIBA) as a harmful microbial metabolite reduced by Pae. HIBA supplementation in drinking water promoted AS inflammation in AS mice. Furthermore, vascular endothelial cells (VECs) were cultured and stimulated by HIBA. We verified that HIBA stimulation increased intracellular ROS levels, thereby inducing VEC inflammation via the TXNIP/NLRP3 pathway. In sum, Pae reduces the production of the microbial metabolite HIBA, thus alleviating the ROS/TXNIP/NLRP3 pathway-mediated endothelial inflammation in AS. Our study innovatively confirms the mechanism by which Pae reduces the harmful metabolites of gut microbiota to alleviate AS and proposes HIBA as a potential biomarker for AS clinical judgment.
Animals ; Mice ; Atherosclerosis/drug therapy* ; Diet, High-Fat ; Endothelial Cells ; Inflammation/drug therapy* ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Reactive Oxygen Species

OBJECTIVE: To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi" (LI 11) and "Zusanli" (ST 36) in the rats with cerebral ischemic reperfusion and the potential mechanism of microglia pyroptosis. METHODS: Sixty SD rats were randomly divided into a sham-operation group, a model group and an EA group, with 20 rats in each group. The Zea Longa method was employed to establish the rat model of the middle cerebral artery occlusion and reperfusion (MACO/R) in the left brain. In the EA group, since the 2nd day of modeling, EA was given at "Quchi" (LI 11) and "Zusanli" (ST 36) of right side with disperse-dense wave, 4 Hz/20 Hz in frequency and 0.2 mA in current intensity, 30 min each time, once a day for lasting 7 consecutive days. The reduction rate of cerebral blood flow was measured with laser Doppler flowmetry during operation. The neurological function of rats was observed using Zea Longa neurobehavioral score. The cerebral infarction volume was detected by TTC staining method. The microglia positive expression in the ischemic side of the cortex was detected with the immunofluorescence method. Under transmission electron microscope, the ultrastructure of cell in the ischemic cortex was observed. The mRNA expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1) and gasdermin D (GSDMD) in the ischemic cortex were detected using real-time PCR. RESULTS: Compared with the sham-operation group, in the model group, the reduction rate of cerebral blood flow was increased during operation (P<0.001); Zea Longa neurobehavional score and the percentage of cerebral infarction volume were increased (P<0.001), the numbers of M1-type microglia marked by CD68⁺ and M2-type microglia marked by TMEM119⁺ were elevated in the ischemic cortex (P<0.001), the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was increased (P<0.001, P<0.01); the cytomembrane structure was destroyed, with more cell membrane pores formed in the ischemic cortex. Compared with the model group, after intervention, Zea Longa neurobehavioral score and the percentage of cerebral infarction volume were reduced (P<0.05), the number of M1-type microglia marked by CD68⁺ was reduced (P<0.05) and the number of M2-type microglia marked by TMEM119⁺ was increased (P<0.05); and the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was decreased (P<0.01, P<0.05) in the EA group. Even though the cytomembrane structure was incomplete, there were less membrane pores presented in the ischemic cortex in the EA group after intervention. CONCLUSION The intervention with EA attenuates the neurological dysfunction and reduces the volume of cerebral infarction in the rats with cerebral ischemic reperfusion. The underlying mechanism is related to the inhibition of microglia pyroptosis through modulating NLRP3/Caspase-1/GSDMD axis.
Animals ; Rats ; Rats, Sprague-Dawley ; Caspase 1/genetics* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Electroacupuncture ; Cerebral Infarction/therapy* ; RNA, Messenger

OBJECTIVE: To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI. METHODS: A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1β and IL-18 in the cerebral cortex of rats were determined by ELISA. RESULTS: Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1β and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1β and IL-18 were higher (P<0.01). CONCLUSION Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; PPAR gamma/genetics* ; Pyroptosis ; Interleukin-18 ; Electroacupuncture ; NLR Family, Pyrin Domain-Containing 3 Protein ; Cerebral Cortex ; Cerebral Infarction/therapy* ; Caspases ; RNA, Messenger

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.
Rats ; Male ; Animals ; Diabetic Nephropathies/genetics* ; Interleukin-18/metabolism* ; Glycosides/pharmacology* ; Tripterygium ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Rats, Sprague-Dawley ; Caspase 1/metabolism* ; Pyroptosis ; Uridine Triphosphate/pharmacology* ; Kidney ; Valsartan/pharmacology* ; RNA, Messenger/metabolism* ; Diabetes Mellitus