Immune and coagulation profiles in 3 adults with multisystem inflammatory syndrome

INTRODUCTION@#There is a paucity of information on the cytokine, complement, endothelial activation, and coagulation profiles of multisystem inflammatory syndrome in adults (MIS-A), a rare but serious complication following recovery from SARS-CoV-2 infection. We aim to examine the immune biomarker and coagulation profiles in association with the clinical presentation and course of MIS-A.@*METHOD@#The clinical features of MIS-A patients admitted to our tertiary hospital were documented. Their levels of interleukin (IL)-1β, IL-6, IL-10, IL-17, IL-18, interferon-α (IFN-α), IFN-γ, interferon gamma-induced protein 10 (IP-10), tumour necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, complement activation product (complement 5a [C5a]), and endothelial biomarker intercellular adhesion molecule-1 (ICAM-1) levels were assayed. The haemostatic profile was assessed with standard coagulation testing and thromboelastography.@*RESULTS@#Three male patients were diagnosed with MIS-A at our centre from January to June 2022 with a median age of 55 years. All had tested positive for SARS-CoV-2 12-62 days prior to MIS-A presentation, with gastrointestinal and cardiovascular systems as the most commonly involved. Levels of IL-6, IL-10, IL-18, IP-10 and MCP-1 were raised whereas IL-1β, IFN-α, IFN-γ, IL-17 and TNF-α remained normal. Markedly elevated levels of C-reactive protein (CRP), ferritin and ICAM-1 were present in all. C5a was elevated in 2 patients. A hypercoagulable state was demonstrated by raised levels of D-dimer, factor VIII, von Willebrand factor antigen, and ristocetin cofactor with corresponding raised parameters in thromboelastography in the 2 patients who had their coagulation profile assessed.@*CONCLUSION@#MIS-A patients demonstrate activation of pro-inflammatory cytokines, endotheliopathy, complement hyperactivation and hypercoagulability.

Effects of electroacupuncture on NLRP3 inflammasome and pyroptosis protein GSDMD in uterine tissue in rats with primary dysmenorrhea / 中国针灸

OBJECTIVE@#To observe the effects of electroacupuncture (EA) on NLRP3 inflammasome and its downstream protein gastermin D (GSDMD) in rats with primary dysmenorrhea (PDM), and to explore the potential mechanism of EA on the treatment of PDM.@*METHODS@#Forty healthy female SD rats without pregnancy were randomly divided into a control group, a model group, an EA group and an ibuprofen group, 10 rats in each group. PDM model was prepared by injection of estradiol benzoate and oxytocin. Except the control group, the rats in each group were subcutaneously injected with estradiol benzoate for 10 days, and oxytocin was injected on the 11th day. The rats in the EA group were intervened with EA (dense wave, frequency of 50 Hz) at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) at the same time of modeling, once a day, 20 min each time, for 10 consecutive days. The rats in the ibuprofen group were treated with 0.8 mL of ibuprofen by gavage (concentration of ibuprofen solution was 1.25 mg/mL) for 10 consecutive days. After modeling, the writhing reaction was observed. After intervention, the HE staining method was used to observe the histological morphology of uterus and evaluate the pathological damage score of uterus; ELISA method was used to detect the serum levels of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α); Western blot method was used to detect the protein expression of NLRP3, apoptosis related spot like protein (ASC), caspase-1, GSDMD, GSDMD-N and inflammatory factors (interleukin [IL]-1β, IL-18) in uterine tissue.@*RESULTS@#In the model group, a large number of vacuolar degeneration and death of endometrial epithelial cells, spiral arterioles congestion in lamina propria and neutrophil infiltration were observed. In the EA group, there was a small amount of vacuolar degeneration and death of endometrial epithelial cells, a small amount of spiral arterioles congestion in the lamina propria, and a small amount of neutrophils infiltration. In the ibuprofen group, there was very small number of degeneration and death of endometrial epithelial cells, and no obvious arterial congestion was found in lamina propria, and neutrophil infiltration was occasionally seen. Compared with the control group, in the model group the number of writhing was increased (P<0.01), the writhing reaction score and serum level of PGF2α and PGF2α/PGE2 value were increased (P<0.01), the level of PGE2 was decreased (P<0.01). Compared with the model group, in the EA group and the ibuprofen group the number of writhing were decreased (P<0.05), the latency of writhing was prolonged (P<0.01), the writhing reaction scores and serum levels of PGF2α and PGF2α/PGE2 values were decreased (P<0.05, P<0.01), the levels of PGE2 were increased (P<0.01). Compared with the control group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 in the uterine tissues of rats was increased in the model group (P<0.01). Compared with the model group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 in the uterine tissues of rats was decreased in the EA group and the ibuprofen group (P<0.01, P<0.05). There was no significant difference between the EA group and the ibuprofen group in the above indexes (P>0.05).@*CONCLUSION@#EA could alleviate pain and uterine tissue injury in rats with PDM. The mechanism may be related to the inhibition of the activation of NLRP3 inflammasome in rat uterine tissues, thereby inhibiting pyroptosis and its inflammatory factors release.

Effect of Shao's five-needle therapy pretreatment on airway inflammatory response in asthmatic rats based on ROS/TXNIP/NLRP3 pathway / 中国针灸

OBJECTIVES@#To explore the possible mechanism of Shao's five-needle therapy pretreatment on relieving airway inflammatory response in asthmatic rats.@*METHODS@#Forty SPF-grade SD rats were randomly divided into a blank group, a model group, an acupuncture group, and a medication group, with 10 rats in each group. Except the blank group, asthma model was established by aerosol inhalation of ovalbumin in the other 3 groups. The rats in the acupuncture group were treated with acupuncture at "Dazhui" (GV 14) and bilateral "Feishu" (BL 13) and "Fengmen" (BL 12), with each session lasting for 20 min. Acupuncture was given before each motivating, once daily for 7 consecutive days. The rats in the medication group were treated with intraperitoneal injection of dexamethasone sodium phosphate solution before each motivating, once daily for 7 days. General situation of the rats was observed in each group; ELISA method was used to detect the levels of inflammatory cytokines interleukin (IL)-1β and IL-18 in serum; immunofluorescence staining method was performed to assess the expression of reactive oxygen species (ROS) in lung tissues; Western blot method was used to measure the protein expression of thioredoxin interacting protein (TXNIP), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in lung tissues.@*RESULTS@#The rats in the blank group exhibited normal behavior, while those in the model group showed signs of respiratory distress, ear scratching, cheek rubbing, and dysphoria. Compared with the model group, the rats in the acupuncture group and the medication group showed stable respiration and relatively agile responses. Compared with those in the blank group, the serum levels of IL-18 and IL-1β were elevated (P<0.01), the expression intensity of ROS was increased, and the protein expressions of TXNIP, NLRP3, ASC and Caspase-1 in lung tissues were increased (P<0.01) in the model group. Compared with those in the model group, the serum levels of IL-18 and IL-1β were reduced (P<0.01), the expression intensity of ROS was lowered, and the protein expressions of TXNIP, NLRP3, ASC and Caspase-1 in lung tissues were reduced (P<0.01) in the acupuncture group and the medication group. Compared with the medication group, the protein expression of ASC in lung tissue was reduced in the acupuncture group (P<0.05).@*CONCLUSIONS@#Pretreatment of Shao's five-needle therapy could alleviate airway inflammatory response in asthmatic rats by reducing ROS levels and decreasing the aggregation and activation of pathway-related proteins in the ROS/TXNIP/NLRP3 pathway, ultimately leading to decreased secretion of IL-1β and IL-18. This mechanism may contribute to the effectiveness of Shao's five-needle therapy in preventing and treating asthma.

Therapeutic effect and mechanism of Xiaoyao Kangai Jieyu Recipe on mice with breast cancer related depression through regulating COX pathway / 中国中药杂志

This study aimed to investigate the intervention effect and mechanism of Xiaoyao Kangai Jieyu Recipe(XKJR) on hip-pocampal microglia and neuronal damage in mice with breast cancer related depression. The mouse model of breast cancer related depression was established by inoculation of 4T1 breast cancer cells in axilla and subcutaneous injection of corticosterone(30 mg·kg~(-1)). The successfully modeled mice were randomly divided into a model group, a positive drug group(capecitabine 60 mg·kg~(-1)+fluoxetine 19.5 mg·kg~(-1)), and XKJR group(19.5 mg·kg~(-1) crude drug), with 6 in each group. Another 6 normal mice were taken as a normal group. The administration groups were given corresponding drugs by gavage, while the normal and model groups were given an equal volume of distilled water, once a day for 21 consecutive days. The depressive behavior of mice was assessed by glucose consumption test, open field test and novelty-suppressed feeding test. Hematoxylin and eosin(HE) staining and tumor suppression rate were used to evaluate the changes of axillary tumors. The mRNA expressions and the relative protein expressions of interleukin-1β(IL-1β), interleukin-18(IL-18), cyclooxyganese-2(COX-2) and glutamyl-prolyl-tRNA synthetase(EPRs) in the hippocampus of mice were determined by quantitative real-time polymerase chain reaction(qRT-PCR) and immunohistochemistry, respectively. Immunofluorescence was performed to detect the mean fluorescence intensity of CD11b, a marker of hippocampal microglia activation. Nissler staining and transmission electron microscopy were employed to observe the morphological changes and the ultramorphological changes of hippocampal neurons, respectively. The experimental results indicated that compared with the normal group, the model group had reduced glucose consumption and lowered number of total activities in open field test(P<0.05, P<0.01), prolonged first feeding latency in no-velty-suppressed feeding test(P<0.01), and significant depression-like behavior; the contents of IL-1β, IL-18, COX-2, and EPRs in hippocampus were increased(P<0.05, P<0.01), with hippocampal microglia activation and obvious neuronal damage. Compared with the model group, the positive drug group and the XKJR group presented an improvement in depressive behaviors, a decrease in the contents of IL-1β, IL-18, COX-2 and EPRs in hippocampus, and an alleviation in the activation of hippocampal microglia and neuronal damage; the tumor suppression rates of positive drug and XKJR were 40.32% and 48.83%, respectively, suggesting a lower tumor growth rate than that of the model group. In summary, XKJR may improve hippocampal microglia activation and neuronal damage in mice with breast cancer related depression through activating COX signaling pathway.

Arctigenin mitigates vascular endothelial injury in rats with pregnancy-induced hypertension via autophagy-NLRP3 inflammasome pathway / 中国中药杂志

This study aims to investigate the effect and mechanism of arctigenin(ARC) in the treatment of vascular endothelial injury in rats with pregnancy-induced hypertension(PIH). Fifty SD rats pregnant for 12 days were randomly assigned into a control group, a model group, an ARC group, a rapamycin(RAP, autophagy inducer) group, and an ARC+3-methyladenine(3-MA, autophagy inhibitor) group, with 10 rats in each group. The rats in the other groups except the control group were intraperitoneally injected with nitrosyl-L-arginine methyl ester(50 mg·kg~(-1)·d~(-1)) to establish the PIH model on the 13th day of pregnancy. On the 15th day of pregnancy, the rats in ARC, RAP, and ARC+3-MA groups were intraperitoneally injected with ARC(50 mg·kg~(-1)·d~(-1)), RAP(1 mg·kg~(-1)·d~(-1)), and 3-MA(15 mg·kg~(-1)·d~(-1))+ARC(50 mg·kg~(-1)·d~(-1)), respectively. The pregnant rats in the control group and the model group were intraperitoneally injected with the same amount of normal saline. The blood pressure and 24 h urine protein(24 h-UP) of pregnant rats in each group were measured before and after intervention. Cesarean section was performed to terminate pregnancy on day 21, and the body weight and body length of fetal rats were compared among groups. Hematoxylin-eosin(HE) staining was employed to observe the pathological changes of placenta. The expression of endothelin-1(ET-1) and endothelial nitric oxide synthase(eNOS) in placenta was detected by immunohistochemistry. The serum levels of ET-1 and nitric oxide(NO) were determined with corresponding kits. The expression of microtubule-associated protein 1 light chain 3(LC3), Beclin-1, NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein with CARD domain(ASC), caspase-1, interleukin(IL)-1β, and IL-18 was determined by immunofluorescence and Western blot. The level of reactive oxygen species(ROS) in placenta was measured by fluorescence staining. The results showed that on day 12 of pregnancy, the blood pressure and 24 h-UP had no significant differences among groups. On days 15, 19, and 21, the blood pressure and 24 h-UP in the model group were higher than those in the control group(P<0.05). On days 19 and 21, the blood pressure and 24 h-UP in ARC group and RAP group were lower than those in the model group(P<0.05), and they were higher in the ARC+3-MA group than in the ARC group(P<0.05). On day 21, the model group had lower body weight and body length of fetal rats(P<0.05), higher serum level of ET-1, and lower serum level of NO(P<0.05) than the control group. Moreover, the placental tissue showed typical pathological damage, down-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and eNOS(P<0.05), up-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1β, and IL-18(P<0.05), and elevated ROS level. Compared with the model group, ARC and RAP groups showed increased body weight and body length of fetal rats(P<0.05), lowered serum level of ET-1, elevated serum level of NO(P<0.05), reduced pathological damage of placental tissue, up-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1, and eNOS(P<0.05), down-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1β, and IL-18(P<0.05), and lowered ROS level. Compared with ARC group, 3-MA reversed the effects of ARC on the above indicators. In conclusion, ARC can inhibit the activation of NLRP3 inflammasome and mitigate vascular endothelial damage in PIH rats by inducing autophagy of vascular endothelial cells.

"Trichosanthis Fructus-Allii Macrostemonis Bulbus" combination inhibits NLRP3 inflammasome activation and reduces inflammatory response in RAW264.7 macrophage by inducing autophagy / 中国中药杂志

This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1β, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1β in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1β, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1β. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.

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.

Terpinen-4-ol inhibits proliferation of VSMCs exposed to high glucose via regulating KLF4/NF-κB signaling pathway / 中国中药杂志

This study aimed to observe the effect of terpinen-4-ol(T4O) on the proliferation of vascular smooth muscle cells(VSMCs) exposed to high glucose(HG) and reveal the mechanism via the Krüppel-like factor 4(KLF4)/nuclear factor kappaB(NF-κB) signaling pathway. The VSMCs were first incubated with T4O for 2 h and then cultured with HG for 48 h to establish the model of inflammatory injury. The proliferation, cell cycle, and migration rate of VSMCs were examined by MTT method, flow cytometry, and wound healing assay, respectively. The content of inflammatory cytokines including interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α) in the supernatant of VSMCs was measured by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to determine the protein levels of proliferating cell nuclear antigen(PCNA), Cyclin D1, KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. The KLF4 expression in VSMCs was silenced by the siRNA technology, and then the effects of T4O on the cell cycle and protein expression of the HG-induced VSMCs were observed. The results showed that different doses of T4O inhibited the HG-induced proliferation and migration of VSMCs, increased the percentage of cells in G_1 phase, and decreased the percentage of cells in S phase, and down-regulated the protein levels of PCNA and Cyclin D1. In addition, T4O reduced the HG-induced secretion and release of the inflammatory cytokines IL-6 and TNF-α and down-regulated the expression of KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. Compared with si-NC+HG, siKLF4+HG increased the percentage of cells in G_1 phase, decreased the percentage of cells in S phase, down-regulated the expression of PCNA, Cyclin D1, and KLF4, and inhibited the activation of NF-κB signaling pathway. Notably, the combination of silencing KLF4 with T4O treatment further promoted the changes in the above indicators. The results indicate that T4O may inhibit the HG-induced proliferation and migration of VSMCs by down-regulating the level of KLF4 and inhibiting the activation of NF-κB signaling pathway.

Effect of Tongdu Tiaoshen electroacupuncture pretreatment on PPARγ-mediated pyroptosis of cerebral cortex in rats with cerebral ischemia reperfusion injury / 中国针灸

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.

CircRNA-SCAF8 promotes vascular endothelial cell pyroptosis by regulating the miR-93-5p/TXNIP axis / 浙江大学学报·医学版

OBJECTIVES@#To investigate the role and mechanism of circRNA-SR-related CTD associated factor 8 (SCAF8) in regulating endothelial cell pyroptosis in high glucose environment.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) were cultured and divided into six groups. The normal control group and high glucose control group were cultured in cell culture medium with 5 and 33 mmol/L glucose, respectively. The RNA control group, circRNA-SCAF8 inhibition group, miR-93-5p overexpression group and miR-93-5p inhibition group were added with non-functional siRNA, circRNA-SCAF8 inhibitor, miR-93-5p overexpression molecule and miR-93-5p inhibitor in high glucose environment, respectively. Cell viability and pyroptosis were detected by cell counting kit-8 (CCK-8) assay, flow cytometry and Hoechst 33342/propidium iodide fluorescence double staining. Western blotting and enzyme-linked immunosorbent assay were used to detect the expression of pyroptosis-related factors including apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartic acid specific protease-1 (caspase-1) and Gasdermin D (GSDMD), NOD like receptor protein 3 (NLRP-3), thioredoxin interacting proteins (TXNIP), IL-18 and IL-1β. The expression of circRNA-SCAF8, miR-93-5p and TXNIP was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fluorescence in situ hybridization (FISH) was used to locate circRNA-SCAF8 and miR-93-5p. Dual luciferase assay was used to verify the targeted regulatory relationship between miR-93-5p and upstream and downstream molecules.@*RESULTS@#Compared with the RNA control group, the cell survival rate of circRNA-SCAF8 inhibition group and miR-93-5p overexpression group increased (both P<0.01), the pyroptosis decreased (both P<0.01), and the expressions of pyroptosis-related factors such as TXNIP, NLRP-3, caspase-1, GSDMD, ASC, IL-18 and IL-1β were significantly decreased (all P<0.05). The expression of miR-93-5p was significantly increased after inhibition of circRNA-SCAF8 (P<0.01), and the expression of circRNA-SCAF8 tended to decrease after overexpression of miR-93-5p, but with no statistical significance (P>0.05). Dual luciferase assay showed that miR-93-5p downre-gulated circRNA-SCAF8 expression by binding to the 3 ´ UTR region of circRNA-SCAF8, and miR-93-5p downregulated TXNIP expression by binding to the 3 ´ UTR region of TXNIP. FISH showed that circRNA-SCAF8 and miR-93-5p were both located in the cytoplasm and were highly associated in the cells. qRT-PCR showed that the relative expression of TXNIP increased or decreased after overexpression or inhibition of miR-93-5p compared with the RNA control group, respectively (both P<0.05), suggesting that miR-93-5p could regulate TXNIP gene expression.@*CONCLUSIONS@#CircRNA-SCAF8/miR-93-5p/TXNIP axis is involved in the regulation of pyroptosis in HUVECs under high glucose.

Upregulation of IL-18 expression in blood CD4+ Th2 cells of patients with allergic rhinitis / 细胞与分子免疫学杂志

Objective To investigate the expressions of IL-18, IL-18 binding protein isoform a (IL-18BPa) and IL-18 receptor α (IL-18Rα) in blood CD4+ Th2 cells of patients with allergic rhinitis (AR) and the effects of allergens on their expressions. Methods Blood samples of AR patients and healthy control subjects (HCs) were collected. Peripheral blood mononuclear cells (PBMCs) and CD4+ T cells sorted by immunomagnetic beads were stimulated by crude extract of Artemisia sieversiana wild allergen (ASWE), Platanus pollen (PPE) and house dust mite extract (HDME). Flow cytometry was used to detect the expression of IL-18, IL-18BPa and IL-18Rα in CD4+ Th2 cells, and BioPlex was used to detect the level of plasma IL-4 and analyze its correlation with the proportion of IL-18+ Th2 cells. Results Compared with HCs, the proportion of IL-18+ cells was increased in Th2 cells of AR patients; MFI of IL-18 was increased, while that of IL-18Rα was decreased. Moreover, allergens induced IL-18 and IL-18Rα expression in sorted CD4+ Th2 cells of HCs and induced IL-18Rα in that of AR patients. Additionally, elevated plasma IL-4 level was found in AR patients, which was moderately correlated with the percentage of IL-18+ Th2 cells. Conclusion Allergens may be involved in the pathogenesis of AR by inducing expression of IL-18 in peripheral blood CD4+ Th2 cells.

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.

Shenfu Injection improves chronic heart failure by regulating pyroptosis based on NLRP3/caspase-1 pathway / 中国中药杂志

This study investigated the mechanisms and targets of Shenfu Injection in the intervention in chronic heart failure(CHF) through the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/caspase-1 signaling pathway. A CHF model was induced in rats by subcutaneous injection of isoproterenol. Model rats were randomly divided into a model group, a Shenfu Injection group, and a MCC950(NLRP3 inhibitor) group, and a blank group was also set up as a control. After 15 days of treatment, echocardiography was performed to measure cardiac function parameters [left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS)]. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of N-terminal pro-brain natriuretic peptide(NT-proBNP), interleukin(IL)-1β, and IL-18. Hematoxylin-eosin(HE) and Masson staining were used to observe morphological changes in myocardial tissues, and Western blot was used to measure the expression levels of NLRP3/caspase-1 pathway-related proteins [NLRP3, caspase-1, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), IL-1β, and IL-18]. The study found that isoproterenol-induced CHF in rats resulted in decreased cardiac function, worsened myocardial fibrosis, increased expression levels of NLRP3, ASC, caspase-1, GSDMD-N, IL-1β, and IL-18 in myocardial tissues, elevated serum inflammatory factors, and induced myocardial cell pyroptosis. Following Shenfu Injection intervention, the Shenfu Injection group showed significantly improved LVEF and LVFS, a significant decrease in NT-proBNP, a marked downregulation of NLRP3, ASC, caspase-1, GSDMD-N, IL-1β, and IL-18 protein expression levels, reduced serum inflammatory factors IL-1β and IL-18 expression in CHF rats, and a decrease in the rate of TUNEL-positive cells. Shenfu Injection can significantly improve cardiac function in CHF, inhibit myocardial fibrosis, and alleviate the progression of myocardial cell pyroptosis through the inhibition of the NLRP3/caspase-1 pathway.

Effect and mechanism of Poria cocos polysaccharides on myocardial cell apoptosis in rats with myocardial ischemia-reperfusion injury by regulating Rho-ROCK signaling pathway / 中国中药杂志

This study aimed to investigate the effect and underlying mechanism of Poria cocos polysaccharides(PCP) on myocardial cell apoptosis in the rat model of myocardial ischemia-reperfusion injury(MI/RI). Male SPF-grade SD rats were randomly divided into a sham group(saline), a model group(saline), low-and high-dose PCP groups(100 and 200 mg·kg~(-1)), and a fasudil group(10 mg·kg~(-1)), with 16 rats in each group. Except for the sham group, the other four groups underwent left anterior descending coronary artery ligation for 30 min followed by reperfusion for 2 h to establish the MI/RI model. The myocardial infarct area was assessed by TTC staining. Histological changes were observed through HE staining. Myocardial cell apoptosis was evaluated using TUNEL staining. Serum lactate dehydrogenase(LDH), creatine kinase MB(CK-MB), interleukin-1β(IL-1β) and IL-18 levels, myocardial superoxide dismutase(SOD) activity and malondialdehyde(MDA) levels were detected by ELISA. Protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved caspase-3, Ras homolog gene A(RhoA), myosin phosphatase target subunit 1(MYPT-1), phosphorylated MYPT-1(p-MYPT-1), and Rho-associated coiled-coil forming kinase 1(ROCK 1) were measured by Western blot. Pathological staining of myocardial tissue revealed that in the model group, there was focal necrosis of myocardial tissue, myocardial cell swelling, unclear boundaries, and neutrophil infiltration. These pathological changes were alleviated in the low-and high-dose PCP groups and the fasudil group. Compared with the model group, the low-and high-dose PCP groups and the fasudil group showed significantly reduced myocardial infarct area and myocardial cell apoptosis rate. Compared with the sham group, the model group exhibited elevated serum LDH, CK-MB, IL-1β and IL-18 levels, increased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and decreased myocardial SOD levels and Bcl-2 protein expression. Compared with the model group, the PCP groups and the fasudil group showed lowered serum LDH, CK-MB, IL-1β and IL-18 levels, decreased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and increased myocardial SOD levels and Bcl-2 protein expression. PCP exhibited a certain preventive effect on myocardial tissue pathological damage and myocardial cell apoptosis in MI/RI rats, possibly related to the inhibition of the Rho-ROCK signaling pathway activation, thereby reducing oxidative stress and inflammatory responses.

Huangdi Anxiao Capsules-containing serum protects cell model from cognitive dysfunction in diabetes via inhibiting NLRP3-mediated pyroptosis / 中国中药杂志

This study aims to investigate the effects and the molecular mechanism of Huangdi Anxiao Capsules(HDAX)-containing serum in protecting the rat adrenal pheochromocytoma(PC12) cells from diabetes-associated cognitive dysfunction induced by high glucose and whether the mechanism is related to the regulation of NOD-like receptor thermal protein domain associated protein 3(NLRP3)-mediated pyroptosis. The PC12 cell model of diabetes-associated cognitive dysfunction induced by high glucose was established and mcc950 was used to inhibit NLRP3. PC12 cells were randomized into control, model, HDAX-containing serum, mcc950, and HDAX-containing serum+mcc950 groups. Methyl thiazolyl tetrazolium(MTT) assay was employed to determine the viability, and Hoechst 33258/PI staining to detect pyroptosis of PC12 cells. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1 beta(IL-1β) and IL-18. Western blot was employed to determine the protein levels of postsynaptic density protein 95(PSD-95), NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), GSDMD-N, and cleaved cysteinyl aspartate specific proteinase-1(caspase-1), and RT-PCR to determine the mRNA levels of NLRP3, ASC, GSDMD, and caspase-1. The immunofluorescence assay was adopted to measure the levels and distribution of NLRP3 and GSDMD-N in PC12 cells. Compared with the control group, the model group showed decreased cell proliferation, increased PI positive rate, down-regulated protein level of PSD-95, up-regulated protein levels of NLRP3, ASC, GSDMD-N, GSDMD, and cleaved caspase-1, up-regulated mRNA levels of NLRP3, ASC, GSDMD, and caspase-1, and elevated levels of IL-1β and IL-18. Compared with the model group, HDAX-containing serum, mcc950, and the combination of them improved cell survival rate and morphology, decreased the PI positive rate, down-regulated the protein levels of NLRP3, ASC, GSDMD-N, GSDMD, and cleaved caspase-1 and the mRNA levels of NLRP3, ASC, GSDMD, and caspase-1, and promoted the secretion of IL-1β and IL-18. The findings demonstrated that HDAX-containing serum can inhibit the pyroptosis-mediated by NLRP3 and protect PC12 cells from the cognitive dysfunction induced by high glucose.

Effects of hydrogen gas on NOD-like receptor protein 3 inflammasomes in the cerebral cortex of rats with traumatic brain injury / 中华危重病急救医学

OBJECTIVE@#To investigate the effect of hydrogen gas on NOD-like receptor protein 3 (NLRP3) inflammasomes in the cerebral cortex of rats with traumatic brain injury (TBI).@*METHODS@#120 adult male Sprague-Dawley (SD) rates were randomly divided into 5 groups (n = 24): sham operation group (S group), TBI model group (T group), TBI+NLRP3 inhibitor MCC950 group (T+M group), TBI+hydrogen gas group (T+H group), TBI+hydrogen gas+MCC950 group (T+H+M group). TBI model was established by controlled cortical impact. NLRP3 inhibitor MCC950 (10 mg/kg) was intraperitoneally injected for 14 consecutive days before TBI operation in T+M and T+H+M groups. 2% hydrogen inhalation was given for 1 hour at 1 hour and 3 hours after TBI operation in T+H and T+H+M groups. At 6 hours after TBI operation, the pericontusional cortex tissues were obtained, the content of Evans blue (EB) was detected to evaluate the permeability of the blood-brain barrier. Water content in brain tissue was detected. The cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL) and the neuronal apoptosis index was calculated. The expressions of Bcl-2, Bax, NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 p20 were detected by Western blotting. The levels of interleukins (IL-1β, IL-18) were detected by enzyme-linked immunosorbent assay (ELISA).@*RESULTS@#Compared with the S group, the content of EB in cerebral cortex, water content in brain tissue, apoptosis index and the expressions of Bax, NLRP3, ASC, caspase-1 p20 in T group were significantly increased, the expression of Bcl-2 was down-regulated, the levels of IL-1β and IL-18 were increased [the content of EB (μg/g): 87.57±6.89 vs. 10.54±1.15, water content in brain tissues: (83.79±2.74)% vs. (74.50±1.19)%, apoptotic index: (62.66±5.33)% vs. (4.61±0.96)%, Bax/β-actin: 4.20±0.44 vs. 1, NLRP3/β-actin: 3.55±0.31 vs. 1, ASC/β-actin: 3.10±0.26 vs. 1, caspase-1 p20/β-actin: 3.28±0.24 vs. 1, Bcl-2/β-actin: 0.23±0.03 vs. 1, IL-1β (ng/g): 221.58±19.15 vs. 27.15±3.27, IL-18 (ng/g): 87.26±7.17 vs. 12.10±1.85, all P < 0.05]. Compared with the T group, the T+M, T+H and T+H+M groups had significant reductions in the content of EB and water content in brain tissue, apoptotic index of the cerebral cortex, the expressions of Bax, NLRP3, and caspase-1 p20 in the brain tissue and the levels of IL-1β and IL-18, significant increases in the expression of Bcl-2. However, there was no significant difference in ASC expression. Compared with the T+H group, the content of EB in the cerebral cortex, water content in brain tissue, and apoptotic index, and the expressions of Bax, NLRP3 and caspase-1 p20 were further down-regulated in T+H+M group, the expression of Bcl-2 was further up-regulated, the levels of IL-1β and IL-18 were further decreased [the content of EB (μg/g): 40.49±3.15 vs. 51.96±4.69, water content in brain tissue: (76.58±1.04)% vs. (78.76±1.16)%, apoptotic index: (32.22±3.44)% vs. (38.54±3.89)%, Bax/β-actin: 1.92±0.16 vs. 2.56±0.21, NLRP3/β-actin: 1.94±0.14 vs. 2.37±0.24, caspase-1 p20/β-actin: 1.97±0.17 vs. 2.31±0.19, Bcl-2/β-actin: 0.82±0.07 vs. 0.52±0.04, IL-1β (ng/g): 86.23±7.09 vs. 110.44±10.48, IL-18 (ng/g): 40.18±3.22 vs. 46.23±4.02, all P < 0.05], but there were no statistical significance in all the indicators between T+M group and T+H group.@*CONCLUSIONS@#The mechanism by which hydrogen gas alleviates TBI may be related to inhibiting NLRP3 inflammasomes in the cerebral cortex of rats.

Naoluo Xintong Decoction activates caspase-1/Gasdermin D pathway to promote angiogenesis of rat brain microvascular endothelial cells after oxygen glucose deprivation/reperfusion injury / 南方医科大学学报

OBJECTIVE@#To investigate the effects of Naoluo Xintong Decoction (NLXTD) on pyroptosis and angiogenesis of brain microvascular endothelial cells (BMECs) and explore the possible mechanisms in rats with oxygen-glucose deprivation/ reperfusion (OGD/R).@*METHODS@#Rat BMECs with or without caspase-1 siRNA transfection were cultured in the presence of 10% medicated serum from NLXTD-treated rats (or blank serum) and exposed to OGD/R. CCK-8 assay, Transwell chamber assay, and tube formation assay were used to assess proliferation, migration, and tube-forming abilities of the cells. The activity of lactate dehydrogenase (LDH) in the culture supernatant was determined using a commercial assay kit, and the levels of inflammatory factors IL-1β and IL-18 were detected with ELISA. The cellular expressions of pro-caspase-1, caspase-1, NLRP3, Gasdermin D, and angiogenesis-related proteins VEGF and VEGFR2 were detected using Western blotting.@*RESULTS@#The BMECs showed obvious injuries after OGD/R exposure. Compared with the blank serum, the medicated serum significantly improved the cell viability, migration ability, and lumen-forming ability (P < 0.01) and lowered the levels of IL-1β and IL-18 and the LDH release (P < 0.01) of the cells with OGD/R exposure. Western blotting showed that in the BMECs exposed to OGD/R, the medicated serum strongly upregulated the expression of VEGF and VEGFR2 proteins (P < 0.01) and reduced the protein expressions of pro-caspase-1, caspase-1, NLRP3, and Gasdermin D (P < 0.01), and transfection of the cells with caspase-1 siRNA further promoted the expressions of VEGFR2 protein in the cells (P < 0.01).@*CONCLUSION@#NLXTD can improve the proliferation, migration, and tube- forming ability and promote angiogenesis of BMECs with OGD/R injury probably by inhibiting the caspase-1/Gasdermin D pathway in pyroptosis, alleviating cell injury, and upregulating the expressions of VEGF and VEGFR2.

Mechanism of n-butanol alcohol extract of Baitouweng Decoction in treatment of vulvovaginal candidiasis based on negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis / 中国中药杂志

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.

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 and clinical significance of NLRP3 inflammasome activated by IL-17A in CRSwNP / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the effects and clinical significance of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activated by interleukin (IL)-17A in chronic rhinosinusitis with nasal polyps (CRSwNP). Methods: Patients underwent nasal endoscopic surgery in the Third Affiliated Hospital of Sun Yat-sen University from January 2020 to December 2021 were collected, including 28 CRSwNP (including 19 males and 9 females, aged 19 to 67 years), 22 chronic rhinosinusitis without nasal polyps (CRSsNP) and 22 controls. qRT-PCR was used to detect the expressions of IL-17A, NLRP3, IL-1β and IL-18 in the three groups, and their correlations were analyzed. The positions of IL-17A, NLRP3 and IL-18 in nasal polys were analyzed by immunofluorescence. Western Blotting and ELISA were employed to detect the expression of NLRP3, IL-1β and IL-18 in the human nasal epithelial cells after using IL-17A stimulation or IL-17A receptor inhibitor. Immunofluorescence was used to observe the NLRP3, IL-1β, and IL-18 protein expression after IL-17A stimulating human nasal epithelial cells, and after the use of IL-17A receptor inhibitor and NLRP3 inhibitor MCC950. The correlations between NLRP3, IL-1β, IL-18 and CT scores, nasal endoscopic scores, visual analogue scale (VAS) scores, and sino-nasal outcome test (SNOT) 22 scores of CRSwNP patients were analyzed. SPSS 20.0 software was used for statistical analysis. Results: The expressions of IL-17A, NLRP3, IL-1β and IL-18 in the tissues of CRSwNP patients were significantly higher than those in CRSsNP group(P=0.018,P<0.001,P=0.005, P=0.016) and the control group(all P<0.001). IL-17A was positively correlated with the expression of NLRP3, IL-1β, and IL-18(r ralue was 0.643,0.650,0.629,respectively, all P<0.05). IL-17A, NLRP3, and IL-18 were co-localized in the epithelial propria of polyp tissue. IL-17A stimulated the expressions of NLRP3, IL-1β, and IL-18 in human nasal epithelial cells. After the use of IL-17A receptor inhibitor, the expressions of NLRP3, IL-1β, and IL-18 were significantly down-regulated. After the use of NLRP3 inhibitor MCC950, IL-17A was significantly down-regulated to promote the expression of NLRP3, IL-1β, and IL-18. The expressions of NLRP3, IL-1β and IL-18 were positively correlated with CT, nasal endoscopy, VAS, and SNOT22 scores in patients with CRSwNP. Conclusions: IL-17A promotes the release of IL-1β and IL-18 by activating the NLRP3 inflammasome and aggravates the severity of the disease in CRSwNP.