| ObjectiveTo evaluate the therapeutic effect and molecular mechanism of Flos Puerariae and Semen Hoveniae extracts(PHE)on DSS induced acute colitis in mice.MethodsAfter decocting and drying the Flos Puerariae and Semen Hoveniae,the PHE were obtained by mixing in a certain proportion,and the chemical constitution in the PHE was analyzed using UPLC-LTQ-Orbitrap-MS/MS.Male C57 BL/6 mice(7 weeks old,18-20g)were randomly divided into four groups(n=8): normal control group(NC),3% DSS intervention group(DSS),low-dose PHE treatment group(PHE-L,185mg/kg/d)and high-dose PHE treatment group(PHE-h,740mg/kg/d).From the 8th day to the 14 th day,mice except NC group freely drank 3% DSS solution.From the 1st day to the 14 th day,Mice in the PHE treatment group were given low dose and high dose of PHE by gavage.The daily body weight and pharmacological reaction were recorded.After drinking DSS on the last day,the mice were killed and colon tissues were collected were taken to be tested.The protective effect of PHE on DSS induced colitis in mice was evaluated by body weight loss,disease activity index(DAI),colon length reduction and intestinal pathological changes(HE staining and immunohistochemistry).Inflammatory cytokines were detected by ELISA and RT-qPCR.Oxidative stress levels(ROS,CAT,SOD,MDA and T-AOC)were detected by biochemical kits.The expression of INOS and COX-2 in mouse colon and the protein expression of JNK,p-JNK,ERK,p-ERK,P38 and p-P38 in MAPK signal pathway were determined by western blotting.16S rRNA sequencing method was used to analyze and explore the improvement of intestinal flora.Results1.Chemical composition of PHEA total of 27 compounds were tentatively identified in PHE,including flavonoids,saponins,volatile oils,alkaloids,sterols,amino acids,Isoflavones and glycoside derivatives constitute most of the bioactive compounds in PHE.2.Therapeutic effect of PHE on DSS induced colitis in miceThe weight of NC group mice was stable and in good condition.The weight of mice in the model group decreased significantly,and the status was poor.The disease activity index also showed the highest value,with bloody stools and rectal bleeding.Compared with NC group,the length of colon was shortened by nearly half.Whereas in the PHE treatment group,the weight loss was attenuated to varying degrees(P<0.01),the disease activity index was effectively reduced(P<0.01),the length of colon was significantly restored(P<0.01),and the pathological damage of colitis tissue in mice was significantly restored(P<0.05).ELISA and biochemical kit were used to detect the expression of inflammatory factors.Compared with NC group,DSS treatment significantly increased the production of NO and PGE2,while PHE administration decreased this inflammatory response(P<0.01).The expression level of proinflammatory factors IL-6 and TNF-α in colon tissue of model group mice was significantly increased(P<0.05),after the administration of PHE,the trend of the above inflammatory factors was reversed,and the difference was statistically significant(P<0.05).Concurrently,the mRNA expression of these proinflammatory factors in colon of model group also increased significantly(P<0.01).PHE administration reduces the expression of mRNA TNF-α 、 IL-6 and IL-1 β(P<0.05),suggesting that the PHE could effectively therapy colitis in mice.3.Effect of PHE treatment on oxidative stress in colitis miceThe contents of ROS and MDA in the model group were more than twice as high as those in the NC group(P<0.01),indicating that there was severe oxidative stress in the colon tissue induced by DSS.after PHE treatment,the contents of ROS and MDA at low and high doses decreased significantly(P<0.01).Simultaneously,the activities of SOD and CAT were significantly decreased in the model group,while the enzyme activity was restored after PHE administration(P<0.05).The decrease of T-AOC in the model group was also ameliorated by PHE treatment(p<0.01).These above data indicate that PHE significantly recovers oxidative stress in colon tissue.4.Effects of PHE treatment on the activation of INOS,COX-2 and MAPK pathways in mice with colitisThe result of Western Blot showed that the expression of INOS and COX-2 in colon samples of DSS group mice was significantly higher than that of NC group mice,but the expression of PHE group mice was lower(P<0.05),p-ERK,p-P38 and p-JNK in DSS group rose notably,and PHE treatment significantly inhibited the protein expression in MAPK signal pathway.5.Effect of PHE on intestinal microbiota compositionSequence analysis of 16S rRNA gene of mouse intestinal flora showed that PHE could change the abundance and composition of intestinal microbiota by enhancing beneficial bacteria such as microbiota verrucosa and reducing harmful bacteria such as proteus,thereby further regulating intestinal immune function to reduce inflammation and achieve the effect of treating acute colitis.ConclusionPHE plays an important role in treating acute colitis by improving the release of inflammatory factors,alleviating oxidative stress,and Adjusting the imbalance of intestinal microbiota.The possible mechanism of PHE in treating acute colitis is realized by blocking the activation of INOS,COX-2 and MAPK signaling pathways to regulate the production of inflammatory cytokines and mediators. |
