Explore The Therapeutic Effect Of Huangqin Tang On Ulcerative Colitis Rats Through PINK1/Parkin Mitochondrial Pathway

ObjectiveTo investigate the effect of Huangqin Tang on mitochondrial autophagy mediated by PINK1/Parkin autophagy pathway in rats with ulcerative colitis(UC)by observing the changes of mitochondrial morphology and expression of autophagy related proteins,so as to explore the effect of Huangqin Tang on NLRP3 inflammasome and futher clarify the mechanism of Huangqin Tang in the treatment of ulcerative colitls model rats.Method1.The rats were weighed and randomly divided into six groups according to body weight,normal group,model group,positive(SASP)group and high,medium,and low dose groups of Huangqin Tang.TNBS compound modeling method was used to establish the rat model of ulcerative colitis by enema.The mental state,body weight,food intake and fecal characteristics of rats before and after treatment were compared,and DAI score was performed to evaluate the effect of Huangqin Tang on the phaysical signs of UC rats.2.Three days after the model was established,the rats were given intragastric administration for seven days.After the last dose,the rats in each group were fasted for 24 hours but given normal drinking water,and colon tissues were taken after being killed.The pathological changes of colon tissues in each group were observed under the naked eye staining and HE staining method,respectively.The establishment of UC model was verify,and score the pathologicai changes in each group to verify the therapeutic effect of Huangqin Tang on UC model rats.3.The blood was collected from the orbit of rats,and the supermatantt was collected after centrifugation.The levels of CAT,MPO,LPO,SOD,GSH-Px and IL-1β、IL-18、TNF-αinflammation in rat serum were detected by Elisa.4.Transmission electron microscope was used to observe the microstructure of mitochondria and the existence of autophagosome and autophagy lysosomes.5.The expression levels of four PINK1,Parkin,LC3,p62 autophagy related proteins and NLRP3 inflammasome were detected by Western blot.6.The gene levels of PINK1,Parkin,LC3,p62 and NLRP3 were detect by real time PCR.Result1.After modeling,the body weight and food intake pf rats in all groups except the blank group decreased,but increased after administration.There was no significant change in the blank group.The results of DAI score showed that the score of model group was significantly higher than that of normal group(P<0.01).And there were different degrees of improvement in each administration group compared with the model group(P<0.01 or P<0.05).2.The pathological changes of colon tissue in each group were observed under naked eyes and microscope respectively.There was no obvious abnormality in the blank group.In the model group,congestion,edema and ulceration of mucosa in different degrees were observed by naked eye.Under microscope,inflammatory cell infiltration,disorder or even disappearance of villus structure,thinning of colonic mucosa and ulceration were found.Compared with the model group,the number of inflammatory cells in the positive drug group and the high and middle dose groups of Huangqin Tang were significantly reduced,the damage of intestinal villus structure was alleviated,and the changes of mucosal ulcer were significantly alleviated(P<0.01).Compared with the model group,the effect of lowdose Huangqin Tang group was not as abvious as other groups,but the comparison results also significant(P<0.05).3.Compared with the normal blank group,the contents of SOD,CAT and GSH-Px in the model group were significantly lower than those in the model group(P<0.01),while the contents of LPO,MPO,TNF-α,IL-1β and IL-18 were significantly higher(P<0.01).Compared with the model group,the positive drug group and middle dose group of Huangqin Tang significantly increased the contents of SOD,CAT and GSH-Px,decreased the contents of LPO,MPO,TNF-α,IL-1β and IL-18(P<0.01);the high dose group of Huangqin Tang significantly increased the contents of SOD,CAT and GSH-Px(P<0.01 or P<0.05),decreased the contents of MPO,IL-1β(P<0.01)and LPO,IL-18,TNF-α(P<0.05),and had no significant effect on the serum levels of SOD,LPO,MPO and IL-18;The lowdose group of Huangqin Tang could increase the content of SOD and GSH-Px(P<0.05)and decreased the level of LPO,IL-18 and MPO(P<0.05),but had no significant effect on the levels of CAT,TNF-α and IL-1β in serum.4.The ultrastructure of mitochondria of rats in each group was observed under transmission electron microscope.It was found that the mitochondrial structure of mitochondria in model group was destroyed,and the phenomenon of mitochondrial autophagy and its unique structure were observed in administration group.5.The results of Western blot showed that the protein expressions of Parkin and p62 and the ratio of LC3Ⅱ and LC3Ⅰ in the model group were lower than those in the blank group,while the expression of PINK1 and NLRP3 was increased,with significant difference(P<0.01 or P<0.05).In each treatment group,the expression of NLRP3 was decreased,while the expression of other indicators was increase.6.The result of Real Time PCR showed that the mRNA levels of PINK1,Parkin,p62 and LC3 in the model group were lower than those in the blank group(P<0.01),while the mRNA expression of NLRP3 was increased(P<0.01).Compared with the model group,the content of NLRP3 mRNA in the tissues of all the treatment groups decreased(P<0.01),and the content of PINK1,Parkin,p62 mRNA in the tissues increased(P<0.01 or P<0.05).the mRNA levels of LC3 in the positive group were significantly increased(P<0.01),but there was no significant change in each dose group of Huangqin Tang.Conclusion1.Huangqin Tang can obviously improve the mental state and pathological changes of colon tissue of ulcerative colitis model rats.2.Huangqin Tang can increased the expression of antioxidant enzymes,reduce the oxidative stress reaction and reduce the content of inflammatory factors in serum of UC model rats.3.Huangqin Tang can increase the protein and gene expression of PINK1,Parkin,LC3 and p62,promote the transformation of LC3 protein to LC3Ⅱ,and reduce the expression of NLRP3 inflammasome.4.The mechanism of Huangqin Tang in the treatment of ulcerative colitis may be ralted to the increase of mitochondrial autophagy,the elimination of excessive peroxide products in the body,alleviating oxidative stress reaction in cells,and the inhibition of the activation of NLRP3 inflammasome in tissues.