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ObjectiveTo study the clinical efficacy of the Qingre Lishi Huazhuo method on patients with chronic gouty arthritis of dampness-heat obstruction syndrome and the effect on nucleotide-binding oligomerization domain-like receptor 3（NLRP3）/interleukin-1β （IL-1β） signaling pathway to preliminarily explore its mechanism. MethodSixty patients with chronic gouty arthritis of dampness-heat obstruction syndrome were enrolled and divided into a treatment group （30 cases） and a control group （30 cases） according to the random number table method. Thirty people were assigned to the healthy group. Patients in the control group were treated with oral Febuxostat， while those in the treatment group were treated with modified Simiaosan combined with Febuxostat. Treatment lasted four weeks. The general clinical data， traditional Chinese medicine （TCM） syndrome scores， serum uric acid （UA）， serum creatinine （SCr）， blood urea nitrogen （BUN）， fasting blood glucose （FPG）， low-density lipoprotein （LDL）， triglyceride （TG）， total cholesterol （TC）， erythrocyte sedimentation rate （ESR）， and C-reactive protein （CRP） of patients were recorded. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of IL-1β，TNF-α， and IL-6，and the levels of NLRP3，cysteinyl aspartate-specific protease-1 （Caspase-1）， and apoptosis-associated speck-like protein containing a CARD （ASC） were detected by Western blot. ResultBefore treatment， the levels of body mass index （BMI）， systolic blood pressure （SBP），diastolic blood pressure （DBP），UA，SCr，BUN，FPG，LDL，TG，and TC in both groups significantly increased （P<0.05，P<0.01），and the levels of HDL significantly decreased as compared with those in the healthy group（P<0.05）. Additionally， the levels of IL-1β， TNF-α， and IL-6 in both groups significantly increased before treatment （P<0.01）. Compared with the results before treatment， patients in the two groups had significant reductions in tube pain， joint tenderness， joint swelling，joint fever， activity disorders， body fatigue， sliminess， bitter mouth， yellow and red urine， and tongue manifestation scores （P<0.05，P<0.01）. Compared with patients in the control group after treatment， those in the treatment group had a significant decrease in joint fever， body fatigue， sliminess， bitter mouth，sticky stool，yellow and red urine， tongue manifestation score， and pulse score （P<0.05）. The total effective rate in the treatment group was 80.0% （24/30）， higher than 56.7% （17/30）in the control group（χ2=11.916，P<0.05）. Compared with the results before treatment， BMI， SBP， DBP， UA， SCr， BUN， FPG， LDL， TG， TC， ESR，CRP， IL-1β， TNF-α， IL-6 levels， and VAS score in both groups significantly decreased （P<0.05，P<0.01）. Compared with patients in the control group after treatment， those in the treatment group had decreased DBP，ESR， IL-1β levels， and VAS score （P<0.05）. Western blot results showed that before treatment， the protein expression of NLRP3， Caspase-1， and ASC in peripheral blood mononuclear cells （PBMCs） of patients in both groups were higher than those in the healthy group （P<0.01）. Compared with the results before treatment， the protein expression of NLRP3， Caspase-1， and ASC in PBMCs in patients of both groups after treatment decreased （P<0.05，P<0.01）. Compared with the control group after treatment， the treatment group showed decreased expression levels of NLRP3 and Caspase-1（P<0.05）. ConclusionThe Qingre Lishi Huazhuo method can effectively improve the clinical symptoms and reduce inflammation of chronic gouty arthritis of dampness-heat obstruction syndrome with good safety. The mechanism may be related to the inhibition of the NLRP3/IL-1β signaling pathway.
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ObjectiveTo investigate the therapeutic effect of Xuebijing injection （XBJ） on sodium taurocholate （Na-Tc）-induced severe acute pancreatitis （SAP） in rats. MethodForty rats were randomly assigned into 5 groups： sham operation group， SAP model group， and low-， medium-， and high-dose （4， 8， 12 mL·kg·d-1， respectively） XBJ groups. SAP model was established by retrograde injection of Na-Tc （1 mL·kg-1） into the biliary and pancreatic ducts. XBJ was injected intraperitoneally 3 days before and 0.5 h after modeling. The ascitic fluid volume and the pancreas weight-to-body weight ratio were measured. The pathological changes of pancreatic tissue were observed via hematoxylin-eosin （HE） staining. The protein levels of formyl peptide receptor 1 （FPR1） and nucleotide-binding oligomerization domain-like receptor 3 （NLRP3） in pancreatic tissue were detected by immunohistochemistry. Western blot was employed to determine the expression levels of NADH-ubiquinone oxidoreductase chains 1-6 （MT-ND1， MT-ND2， MT-ND3， MT-ND4， MT-ND5， and MT-ND6） in rat plasma. ResultCompared with sham operation group， the SAP model group showcased increased ascitic fluid volume and pancreas weight-to-body weight ratio （P<0.05）， serious lesions in pancreatic tissue， increased total pathological score （P<0.05）， and up-regulated protein levels of FPR1 and NLRP3 in pancreatic tissue （P<0.05）. The model group had lower MT-ND2 level （P<0.05） and higher MT-ND1， MT-ND3， and MT-ND6 levels in plasma （P<0.05） than the sham operation group， while MT-ND4 and MT-ND5 had no significant differences between the two groups. Compared with SAP model group， the XBJ treatment decreased ascitic fluid volume and pancreas weight-to-body weight ratio （P<0.01）， ameliorated pancreatic lesions， and down-regulated the protein levels of FPR1 and NLRP3 in pancreatic tissue （P<0.01）. The treatments， especially high-dose XBJ （P<0.01）， down-regulated the expression of MT-ND1 （P<0.01）， MT-ND3 （P<0.01）， MT-ND6 （P<0.01）， and MT-ND4 and did not change that of MT-ND5. ConclusionXBJ may antagonize partial mitochondrial N-formyl peptides and excessive inflammatory response mediated by FPR1/NLRP3 to treat SAP in rats.
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OBJECTIVE@#To observe the effect of pretreatment of acupuncture on the expression of nucleotide-binding oligomerization domain-like receptor 3(NLRP3), Caspase-1, interleukin1β(IL-1β) and the number of activated microglia (MG) in the hippocampus in Alzheimer's disease (AD) like rats, so as to explore the mechanism of pretreatment of acupuncture in preventing and treating AD.@*METHODS@#A total of 36 SD rats were randomly divided into a blank group, a model group and an electroacupuncture (EA) group, 12 rats in each group. The AD like rat model was established by 8-week continuous intraperitoneal injection of D-galactose (120 mg·kg@*RESULTS@#Compared with the blank group, the average escape latency was prolonged (@*CONCLUSION@#Pretreatment of acupuncture could prevent and treat the learning-memory dysfunction in AD like rats, and its mechanism may be related to the inhibition of NLRP3 inflammatsome related protein and MG activation.
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Animals , Rats , Alzheimer Disease/therapy , Electroacupuncture , Hippocampus , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Rats, Sprague-DawleyABSTRACT
Objective::To investigate the protective effect of modified Yinchenhao Tang on α-isothiocyanate(ANIT)-induced cholestatic liver disease (CSLD). Method::Wistar rats were randomly divided into 7 groups: blank control group, model control group, compound Glycyrrhizin capsules group(22.5, 45 mg·kg-1), modified Yinchenhao Tang low, middle and high dose groups(4.1, 8.1, 16.2 g·kg-1). A model of cholestatic liver injury was prepared by intragastric administration of ANIT (100 mg·kg-1). Glycyrrhizin capsules and modified Yinchenhao Tang were administered intragastrically on the second day of modeling for 4 consecutive days. And bile duct intubation was performed on the fifth day to measure the bile flow rate of the rats, and serum was taken to test the total bilirubin(TBIL), direct bilirubin(DBIL), indirect bilirubin(IBIL), alanine aminotransferase(ALT) and total bile acid(TBA) serological indicators of each group. Pathological changes of liver tissues were observed by hematoxylin-eosin (HE) staining. The expression levels of G protein-coupled bile acid receptor(TGR5), nucleotide binding oligomerization domain-like receptor 3(NLRP3) and cysteinyl aspartate specific proteinase-1(Caspase-1) proteins in the iver tissues were detected by Western blot. Result::Compared with the blank control group, bile flow rate in the model group decreased significantly(P<0.01). TBIL, DBIL, IBIL, ALT and TBA level in serum were significantly increased(P<0.01), liver tissue lesions were severe, and significantly increased the expression of liver tissue TGR5 and Caspase-1.Compare with model group, the compound Glycyrrhizin capsules group had no significant effect on bile flow rate and TBIL, DBIL, IBIL, ALT and TBA level in serum. Bile flow rate increased and TBIL, DBIL, IBIL, ALT and TBA level in serum decreased significantly in modified Yinchenhao Tang high dose group. The compound Glycyrrhizin capsules group and modified Yinchenhao Tang group have different extents of improvement the pathological changes of the lung tissues, and the protein expression of TGR5 and Caspase-1 were significantly decreased in the liver tissue(P<0.01). Conclusion::Modified Yinchenhao Tang can effectively treat CSLD in rats, and its mechanism may be related to bile acid and bile acid receptor TGR5-mediated inflammatory factors.
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Objective: To investigate the therapeutic effect and related mechanism of Shenling Baizhu San on 3% dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mice. Method: Sixty male SPF C57BL/6 mice were randomly divided into control group, salazosulfapyridine (SASP, 0.52 g·kg-1), and low, medium and high-dose (31.2, 15.6, 7.8 g·kg-1) Shenling Baizhu San groups. Except for the control group, mice in the other groups were given distilled water containing 3% dextran sulfate sodium salt for a week to establish UC models. The drug was administered once a day for 14 days. The normal group and the model group were administered with 0.9%physiological saline at 20 mL·kg-1. The mice's body weight, fecal traits, and occult blood were observed daily, and the disease activity index (DAI) was scored. After the end of the administration, the blood was collected, mice colons were collected, weighed and measured for length, and pathological sections were prepared. Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the serum levels of interleukin-18 (IL-18) and IL-1β in mice; htoxylin eosin (HE) and alixin blue/schiff periodic acid shiff(AB/PAS) staining were used to observe the pathological changes of colon tissues; Western blot was used to detect the colon tissue of mice nucleotide binding oligomerization domain-like receptor 3 (NLPR3), NLPR6 protein expression levels. Result: Compared with the normal group, the DAI score of the model group was increased (PPβ content increased (PPPβ concentration was decreased (PPPPConclusion: Shenling Baizhu San has the effect in treating DSS-induced UC mice, which may be related to the regulation of NLRP3, NLRP6 protein and related inflammatory factors, so as to reduce intestinal inflammation and alleviate intestinal mucosal damage.
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Objective@#To investigate the interventional effects of BAY11-7082 on lung inflammatory response at the early stage and acute lung injury of rats with severe burns.@*Methods@#(1) Experiment 1. Twelve Sprague-Dawley (SD) rats were divided into control (C) group and burn (B) group according to the random number table, with 3 rats in group C and 9 rats in group B. Rats in group C did not receive any special treatment. Rats in group B were inflicted with 30% total body surface area full-thickness burn on the back. Immediately after injury, rats in group B were intraperitoneally injected with normal saline in the dosage of 50 mL/kg. Abdominal aorta blood and lung tissue samples were collected from three rats in group B at post injury hour (PIH) 12, 24, and 48, respectively. The interleukin-1β (IL-1β) and the IL-18 content of serum were determined with enzyme-linked immunosorbent assay. The mRNA expressions of IL-1β and IL-18 in lung tissue were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR). Sample collection and determination in rats of group C were performed as above. (2) Experiment 2. Eighteen SD rats were divided into control (C) group, simple burn (SB) group, and BAY11-7082 intervention (BI) group according to the random number table, with 6 rats in each group. Rats in group C did not receive any special treatment. Rats in groups SB and BI were inflicted with injury as in experiment 1. Immediately after injury, rats in group SB were intraperitoneally injected with normal saline in the dosage of 50 mL/kg, and those in group BI with 8 mg/mL (final mass concentration) BAY11-7082 solution in the dosage of 50 mL/kg. Lung tissue and bronchoalveolar lavage fluid (BALF) of rats with burns were collected at the optimal observation time point concluded from experiment 1. The morphology of lung tissue was observed with hematoxylin-eosin staining, and the pathological damage of lung tissue was graded. The myeloperoxidase (MPO) content of lung tissue and the total protein content of BALF were detected by microplate reader. The protein expressions of nucleotide-binding oligomerization domain-like receptor-3 (NLRP3) and cysteine-aspartic proteases 1 (caspase-1) in lung tissue were determined with Western-blotting. The mRNA expressions of IL-1β, IL-18, NLRP3, and caspase-1 in lung tissue were determined with real-time fluorescence quantitative RT-PCR. Sample collection and determination in rats of group C were performed as above. Data were processed with one-way analysis of variance and LSD-t test.@*Results@#(1) The IL-1β and IL-18 content of serum in rats of group B at PIH 12, 24, and 48 were significantly higher than those of group C (t=10.55, 22.05, 12.47, 10.60, 15.22, 11.94, P<0.01). The mRNA expressions of IL-1β and IL-18 in rats of group B at PIH 12, 24, and 48 were significantly higher than those of group C (t=3.62, 7.19, 5.28, 3.20, 12.62, 7.31, P<0.05 or P<0.01). PIH 24 was the optimal observation time point for the following experiment. (2) At PIH 24, compared with those in group SB, the inflammatory cell infiltration and erythrocyte exudates of alveolar in group BI were obviously reduced, and the pulmonary interstitial edema obviously subsided. The pathological damage score of lung tissue in rats of group SB was (9.00±1.00) points, significantly higher than (1.10±0.26) points of group C (t=13.23, P<0.01). The pathological damage score of lung tissue in rats of group BI was (4.93±0.70) points, which was significantly lower than that of group SB (t=5.76, P<0.01) but still significantly higher than that of group C (t=8.84, P<0.01). At PIH 24, the MPO content of lung tissue and the total protein content of BALF in rats of group SB were (1.83±0.15) U/mg and (1.39±0.20) mg/mL, respectively, significantly higher than (0.51±0.10) U/mg and (0.44±0.05) mg/mL of group C (t=12.50, 7.86, P<0.01). The MPO content of lung tissue and the total protein content of BALF in rats of group BI were (0.91±0.12) U/mg and (0.60±0.10) mg/mL, respectively, significantly lower than those of group SB (t=8.36, 6.06, P<0.01). At PIH 24, the protein expressions of NLRP3 and caspase-1 in lung tissue of rats of group SB were 3.10±0.09 and 2.99±0.30, respectively, significantly higher than 1.00 and 1.00 of group C (t=9.06, 11.28, P<0.01). The protein expressions of NLRP3 and caspase-1 in lung tissue of rats of group BI were 1.13±0.08 and 1.81±0.11, respectively, significantly lower than those of group SB (t=7.24, 3.91, P<0.05 or P<0.01). At PIH 24, the mRNA expressions of IL-1β, IL-18, NLRP3, and caspase-1 in lung tissue of rats in group SB were 5.0±0.4, 3.32±0.21, 3.54±0.42, and 6.3±1.0, respectively, significantly higher than 1.0, 1.00, 1.00, and 1.0 of group C (t=13.97, 14.14, 11.78, 7.13, P<0.01). The mRNA expressions of IL-1β, IL-18, NLRP3, and caspase-1 in lung tissue of rats in group BI were 2.6±0.5, 2.00±0.28, 1.39±0.21, and 2.5±0.5, respectively, significantly lower than those of group SB (t=7.11, 5.80, 9.99, 4.65, P<0.05 or P<0.01).@*Conclusions@#Applying BAY11-7082 at the early stage of acute lung injury of rats with severe burn can reduce the expression of caspase-1, decrease the levels of IL-1β and IL-18, and decrease the MPO content of lung tissue and the total protein content of BALF through inhibiting NLRP3, thus alleviating the lung inflammatory response and lung injury.
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Objective To investigate the role of purinergic 2X7 receptor (P2X7R) and its downstream target-NLRP3 inflammasome activation in the process of pancreatic fibrosis in a mouse model of chronic pancreatitis (CP). Methods Forty C57BL/6 mice were randomly divided into normal control group, CP group, P2X7R antagonist oxidized ATP (OxATP) group and brilliant blue G (BBG) group. The chronic pancreatitis model was induced by repeated intraperitoneal injection of the cholecystokinin analogue caerulein with the dose of 50μg/kg for six weeks. Normal saline, OxATP (20μL, 300μmol/L) or BBG (20μL, 10μmol/L) were administered for CP group, OxATP group and BBG group for two weeks after the last caerulein injection. Then all mice were sacrificed and the histopathological changes of the pancreas, especially the fibrotic degrees were evaluated by HE stain, fibrosis score, Sirius red staining and α-SMA immunohistochemical stain. The pancreatic P2X7R, NLRP3 and Caspase-1 expressions were detected by immunohistochemistry respectively to compare the changes between the groups, and explore the role of P2X7R-NLRP3 signaling pathway in pancreatic fibrosis. Results Compared with the normal control group, the scores of pancreatic fibrosis and the expressions of P2X7R, NLRP3 and Caspase-1 in pancreas were significantly increased in CP model group (P<0.05). Compare to CP group, the pancreatic chronic inflammation and the fibrosis indices such as HE fibrosis score, Sirius red staining and α-SMA immunohistochemical stain were ameliorated obviously in OxATP and BBG groups (P<0.05). And expressions of P2X7R, NLRP3 and Caspase-1 in the pancreas were allreduced greatly in both OxATP and BBG groups (P<0.05). Conclusion P2X7R antagonist OxATP and BBG can significantly decrease pancreatic chronic inflammation and fibrosis in the mouse model of CP, which suggests that the blockade of P 2X7R-NLRP3 inflammasome signaling pathway may represent a novel therapeutic strategy for CP and its fibrotic process.