Pharmacodynamic Ingredients Of Huangqin Decoction Protects Mice Against DSS-induced Colonic Inflammation Via Inhibiting NLRP3 Inflammasome

Ulcerative colitis(UC)is one of the serious gastrointestinal diseases.Recently,the incidence rate of UC increased gradually in our country,however,the therapy of UC is a serious problem.Huangqin decoction is a famous ancient recipe for clearing away heat,stopping dysentery and relieving pain.In clinical,it is used for curing acute gastroenteritis,bacterial dysentery,UC and other gastrointestinal diseases.However,like other traditional Chinese medicines,due to the complex composition and unclear targets,it is difficult to elucidate the therapeutic mechanism for this disease.UC is a series of abnormal immune response caused by intestinal inflammatory response.As the core of inflammatory reactions,NLRP3inflammasome plays an important role in the development of UC.In order to explore its therapeutic effects in depth mechanism,this study used DSS-induced colitis as an animal model in vivo and RAW264.7 cells stimulated by LPS as an inflammation model in vitro to explore the mechanism of pharmacodynamic ingredients of Huangqin Decoction.Objective:The main purpose of this study is to evaluate the therapeutic effects of Huangqin Decoction performs on UC mice by observe the body weight changes of the mice and pathological changes of colonic mucosa.Furthermore,this study would like to find one or several pharmacodynamic ingredients in Huangqin decoction,which showed strong targeted colonic activities and illustrate their mechanism of the therapeutic effect on UC.Methods:1.Therapeutic effect of Huangqin Decoction on DSS-induced ulcerative colitis miceSixty C57BL/6 mice were divided into six groups randomly,including normal group,model group,sulfasalazine group and Huangqin Decoction group.Except for the normal group,other groups were drink 3%DSS freely for 7 days.On day1 to day 10,RO water,sulfasalazine and Huangqin decoction were given by intragastric administration respectively.Weight changes and stool status were recorded daily.On the 11th day,the mice were sacrificed and their tissues such as peripheral blood,thymus and spleen were collected.The spleen was weighed to calculate the index of spleen and detect the ratio of monocyte cells in peripheral with the automatic blood analyzer.2.Disturbution of Baicalin,Wogonoside,Glycyrrhizin,and Paeoniflorin in tissuesBased on the previous study on the effects of Huangqin decoction on the UC mice,the main components of(baicalin,wogonoside,glycyrrhizin,glycyrrhizin,and paeoniflorin)distribution in tissue were determined by UPLC-Q-TOF-MS.Firstly,establishe the liquid mass spectrometry conditions and the standard curve were detected by wild type mice.The content of paeoniflorin in the tissue was detected by negative ion mode,others were detected by positive ion mode.The content of each monomer in the tissues at different time points(0.5h,1h,3h,12h)was calculated according to the standard curve.3.Wogonoside Protects Mice against DSS-Induced Colonic Inflammation via Inhibiting NLRP3 Inflammasome3.1 Therapeutic effect of wogonoside on UC miceTo observe therapeutic effect of wogonoside on UC mice,body weight,colon length,organ index,histological and the number of peripheral leukocytes were analyzed.3.2 Wogonoside protect DSS-induced colitis inflammation of UC miceAfter administrated with Wogonoside,UC mice were sacrificed and the colon and fixed sections were obtained.The macrophages in the colon were labeled by F4/80~+marker and the colon macrophages were detected by immunohistochemistry.3.3 Effect of Wogonoside on UC mice via inhibiting the NLRP3 inflammasome formation and activationThe colon F4/80~+macrophages were co-localized with NLRP3/Caspase-1 and NLRP3/ASC and detected by immunofluorescence.When NLPR3 inflammasome formed and activated,IL-1βand IL-18 increased.These cytokines in the supernatant of colon tissue were detected by ELISA kit.3.4 Wogonoside effects on LPS-stimulated RAW264.7 cellsRAW264.7 cells stimulated by LPS as a classic inflammation model,adding certain amounts of wogonoside for intervention,the LPS-stimulated RAW264.7cell activity was detected by MTT,which determined the non-cytotoxic and effective dose.The effect of wogonoside on phagocytosis of RAW264.7 cells was detected by flow cytometry.3.5 Effect of Wogonoside on TXNIP,iNOS and p-p65 protein in LPS-stimulated RAW264.7 cellsLike the previous operation,cytoplasmic proteins were extracted on the RAW264.7 cells after LPS stimulation for 24h,western blot was used to detect the expression of TXNIP,iNOS and p-p65 protein.3.6 Effect of Wogonoside on the secretion of NO and TNF-αThe model of RAW264.7 cells induced by LPS was adding certain dose of wogonoside and the LPS stimulated for 24 hours,then the cells supernatants were collected and the NO detection kit was used to detect the changes of NO secretion.CBA detection kit detected the cytokine of TNF-α.3.7 Effect of Wogonoside on the formation of NLRP3 inflammasome in LPS-stimulated RAW264.7 cellsAfter LPS stimulation for 24h,co-localization of NLRP3 and ASC,NLRP3and Caspase-1 in RAW264.7 cells was detected by laser scanning confocal microscope,which detected the effect of wogonoside on the formation of NLRP3inflammasome in RAW264.7 cells.3.8 Effect of Wogonoside on NLRP3 inflammasome activation in LPS-stimulated RAW264.7 cellsCytoplasmic proteins were extracted and the expressions of NLRP3,ASC and Caspase-1 were detected by western blot.Results:1.Therapeutic effects of Huangqin Decoction on DSS-induced ulcerative colitis miceWhen given the mice with 3%DSS for 7 days,which would accompany by the symptoms of weight loss,dark dullness and contracture.Colon become shorter,HE staining showed that the colon ulcer lesions involving the intestinal mucosa,the part of the colon lamina propria disappeared,mucosal edema thickening and necrosis of epithelial cells shedding.Compared with the normal group,the proportion of monocytes and neutrophils in peripheral blood was significantly increased(P<0.01 or P<0.001).Those indicators have recovered by sulfasalazine.Compared with the model group,the body weight of mice decreased slowly(P<0.05),while the ratio of monocytes and neutrophils in leukocytes decreased especially of the medium and high dose group(P<0.01).Huangqin Decoction diminished the DSS-induced massive colonic infiltration of macrophages.All those indicated Huangqin decoction protects on DSS-induced ulcerative colitis mice.2.Distribution of Baicalin,Wogooside,Glycyrrhizin,Glycyrrhizinate and Paeoniflorin in tissues.After the normal mice were orally with Huangqin decoction at the highest dose,the mice were sacrificed at 0.5h,1h,3h and 12h respectively and the heart,liver,kidney and colon were taken from the mice.Wogonoside are highest in the heart,liver and kidney and the content of baicalin in colon was the highest,followed by Wogonoside.However,when we analyzed the relative content of wogonoside(the colon concentration relative to that in HQT),the ratio of absorption of wogonoside into the colon was higher than other compounds.3.Wogonoside ameliorated DSS-Induced colitis inflammation attack in mice via inhibiting the NLRP3 inflammasome3.1 Wogonoside ameliorated DSS-Induced colitisWogonoside treatment attenuated the body weight loss(percentage of that on day 1)compared with the model group,DSS caused colonic shortening,while the symptom was markedly improved by wogonoside treatment during colitis progression.We also measured the spleen index in each group,which was raised in the model group but reduced upon administration of sulfasalazine or wogonoside.Wogonoside also significantly increased the white blood cells(WBC)count in peripheral blood.In particular,the ratios of monocytes to lymphocytes were significantly different between the model group and treatment groups.Colonic inflammation and ulceration were evaluated by histopathological analysis using hematoxylin and eosin staining and a histological score was determined by a standard method.Mucosal inflammation,missing epithelium and submucosal infiltration were detected in colon tissues from the model group.On the opposite,wogonoside relieved the severe symptoms in the colon remarkably.3.2 Wogonoside ameliorated DSS-induced colitis inflammation attack in miceTo assess the extent of intestinal in fl ammation in the colons of the DSS-treated mice,colon tissue sections were stained with an F4/80 antibody to detect macrophage infiltration,the data indicated that wogonoside was able to suppress the massive colonic infiltration of macrophages,thus confirming the anti-inflammatory effect of wogonoside.3.3 Wogonoside lessened acute colitis by inhibiting the NLRP3inflammasomeThe results showed that the colocalization of red and green fluorescence in the mice of the model group was significantly higher than the normal group,and the expression of NLRP3 and Caspase-1 protein in the colonic macrophages of the mice in the durg treatment group was significantly reduced.In the presence of stimuli,the formation of NLRP3 inflammasome causes an increase in the secretion of IL-1βand IL-18,which acts as an effector molecule for the formation and activation of NLRP3 inflammasomes by detecting IL-1 in colon homogenates.The content of IL-1βand IL-18 in DSS group increased significantly(P<0.01).Wogonoside decreased the content of IL-1βand IL-18in colon tissue(P<0.05).3.4 Effects of wogonoside on RAW 264.7 cells upon LPS stimulationCell viability was evaluated using the MTT method to assess the cytoprotective effects of wogonoside on the growth of RAW 264.7 cells.Obviously,the LPS concentration used affected the cell proliferation compared with that in the control group,whereas wogonoside(12.5,25,50μM)could change the proliferation rate of RAW264.7 cells.To assess whether wogonoside could inhibit the LPS-induced inflammation,we analyzed the phagocytosis ability of RAW 264.7 cells.FACS analysis showed a significantly decreased phagocytic rate when the cells were treated with wogonoside.3.5 The effect of wogonoside on the protein of TXNIP、iNOS and p-p65in LPS stimulated RAW264.7 cellThe protein of TXNIP was significantly decreased in model group compared with the normal group(P<0.05).However,when the macrophages were treated with wogonoside,the protein restored to the normal level in a concentration-dependent manner.The expression of iNOS,which were increased in model group and suppressed by the Wogonoside in a concentration-dependent manner.The p-p65 level expression markedly up-regulated in the model group and was inhibited by wogonoside.3.6 Effect of wogonoside on NO and TNF-αin LPS-stimulated RAW264.7 cellsWogonoside inhibited NO secretion(P<0.01)and also inhibited TNF-αsecretion in a dose-dependent manner.3.7 Effect of wogonoside on the formation of NLRP3 inflammasome in LPS-stimulated RAW264.7 cellsConfocal showed that the red and green fluorescence co-localized yellow fluorescence when the Caspase-1 and NLRP3 proteins were co-localized.Compared with the normal group,co-localization of NLRP3 and Caspase-1 in the model group increased significantly(P<0.001),and the intensity of yellow fluorescence decreased significantly in the drug administration groups(P<0.05 and P<0.01).Co-localization of NLRP3 and ASC proteins yielded present similar results.3.8 Wogonoside inhibited LPS-induced NLRP3 expression and activation in RAW264.7 cellsWe further explored whether the wogonoside protective effect was due to the inhibition of NLRP3 inflammasome activation in vitro.We measured the protein level of NLRP3 in the cytoplasm of RAW 264.7 cells.Interestingly,the NLRP3 protein increased in the model group and was significantly suppressed by wogonoside treatment in a concentration-dependent manner.And the ASC protein was measured,which were not changed in any group.Importantly,the activation of Caspase-1(the equal amounts of the cleaved and pro-enzyme forms)was significantly inhibited by wogonoside.Conclusion:1.Huangqin decoction ameliorated DSS-induced colitis;2.The Baicalin,Wogonoside,Glycyrrhizin,Glycyrrhizinate and Paeoniflorin were detected in tissues and the ratio of absorption of wogonoside into the colon was higher than that of the other compounds;3.Wogonoside protects mice against dextran sulfate sodium-induced colonic inflammation via inhibiting NLRP3 inflammasome in a“TXNIP-iNOS-NLRP3”dependent manner.