Study On The Effect Of Kuijieankang Decoction On Intestinal Mucosa Of Rats With Ulcerative Colitis By Regulating PI3K-AKT Signaling Pathwa

Purpose:This study aims to further explore the mechanism of Kuijie Ankang Decoction in the treatment of TNBS/ ethanol-induced UC model rats on the basis of previous experiments.So as to reveal whether Kuijie Ankang Decoction can further inhibit intestinal inflammation,improve local intestinal hypoxia environment,inhibit pathological vascular hyperplasia,reduce intestinal mucosal cell atrophy and other aspects by regulating PI3K/AKT signaling pathway in UC model rats,and ultimately achieve the purpose of UC treatment.Materials and Methods: In this experiment,Fifty-two Sprague-Dawley(SD)male rats with body weight of(200+20)g were purchased from Liaoning Experimental Animal Center.The qualification certificate rats were fed in an air-conditioned room with room temperature(20±2)℃ and relative humidity of 50%-60%,which was approved by the Ethics Committee of Liaoning University of Traditional Chinese Medicine.Fifty-two male SD rats were randomly divided into four groups according to body weight by random number table method:normal group,model group,Kuijie Ankang decoction group,sulfapyridine,sulfapyridine.SASP)group with 13 rats in each group,The model rats with ulcerative colitis were constructed by the mixed solution of trinitrobenzene sulfonic acid(TNBS)and ethanol.After21 days of treatment,the DAI of each group was scored.The pathological smears stained with HE staining of rat colon tissue were observed under electron microscope.The enzyme-related immunosorbent assay was adopted.The serum concentration of NO in UC rats was detected by ELISA,and the colon tissue of UC rats was detected by reverse transcription PCR(RT-PCR).RT-PCR was used to detect the expression levels of PI3 KmRNA and AKTmRNA in UC rats,and Western blot was used to detect the expression levels of p-AKT protein in colonic mucosa of rats to analyze the mechanism of Kuijieankang Decoction in treating UC by regulating PI3K/AKT signaling pathway.Results:1.In general situationThe rats in the normal group had normal mental activity,glossy hair,normal stool characteristics,normal appetite,and weight gain.After inflammation,the rats in the model group had listless spirit,dark hair,watery stool,blood in stool,loss of appetite,and obvious decrease in body weight.After treatment with sulazopyridinium(SASP)and Kuijie Ankang Decoction,the activity,hair color,blood in stool,appetite and body weight of rats in the two groups were significantly improved compared with the model group.2.Influence of Kuijie Ankang Decoction on DAI score of UC model ratsBy calculating DAI scores of rats,it was found that DAI scores of rats in the model group were significantly higher than those in the normal group,the difference was statistically significant(P<0.01);Compared with model group,DAI scores of Kuijie Ankang Tang group and SASP group were decreased,and the difference was statistically significant(P<0.01);DAI score of Kuijie Ankang Decoction group decreased significantly on day 1,day 7 and day 21,and there was no significant difference compared with SASP group(P>0.05).3.The effect of Kuijie Ankang Decoction on colonic morphology of UC model rats was observed by naked eye.In the normal group,no obvious abnormalities were observed in the colon tissue from rectum to ileocecal end,the intestinal cavity was unobstructed,the intestinal wall was evenly thin and thick,no erosion ulcers were observed,the mucosa was smooth,no edema and congestion were observed,and the plicae were complete and clear.In the model group,erosion and large and deep ulcerous surface were observed in the colon tissue,the damaged intestinal wall was hard and locally thickened,black and brown,a small amount of fecal mass retention was observed in the proximal colon,and obvious congestion,blood stasis and edema were observed in the local colon mucosa.No obvious erosion or ulceration was observed in the colonic mucosa of UC model rats after Kuitiangantang and SASP treatment.The color of the colonic mucosa was close to that of the normal group,and the plicae were complete and clear.Only a small amount of mucosal congestion and edema was observed 8cm away from the anus.4.The colonic tissue of UC model rats was observed by Kuijie Ankang Decoction under the pathological HE staining microscopeIn the normal group,the colonic mucosa epithelium was intact,the texture was clear,and the glands were arranged regularly.The morphology of the inherent intramembrane goblet cells was normal,and no inflammatory cell infiltration and small ulcers were observed.In the model group,the ulcer depth of colonic lesion could reach the mucous muscle layer,the crypt cells were lost,the epithelial cells were exfoliated and necrotic.Lamina propria glands are disordered,atrophied or even completely lost,and goblet cells are reduced.A large number of inflammatory cells infiltrated the mucosa and submucosa,mainly neutrophils invaded the whole layer,and the continuity of the mucosa was obviously damaged;after the intervention of Kuitiangang Decoction and sulfasopyridine(SASP),the inflammatory cell infiltration of colon tissue in UC model rats was significantly reduced compared with the model group,and a small amount of glandular canal distortion was observed.The degree of mucosal inflammatory cell infiltration,glandular structure disorder,epithelial cell loss and ulcer degree were improved to varying degrees.5.Effect of Kuijie Ankang Decoction on serum NO content in UC model rats.Compared with normal group,the serum NO content in model group was significantly increased,and the difference was statistically significant(P< 0.01).The serum NO content in all treatment groups was lower than that in model group,and the difference was statistically significant(P< 0.01).There was NO significant difference in the content of NO in serum between Kuijie Ankang Decoction group and SASP group(P>0.05).6.Effect of Kuijie Ankang Decoction on VEGF mRNA expression in colon tissues of UC model rats.Compared with normal group,VEGF mRNA in colon tissue of rats in model group was significantly increased,with statistical significance(P< 0.01).Compared with model group,the relative expression level of VEGF mRNA in colon tissue of rats in treatment groups was significantly decreased,with significant statistical significance(P<0.01).There was no significant difference in the relative expression level of VEGF mRNA in colon tissues of Kuijie Ankang Decoction group compared with SASP group(P<0.01).7.Effect of Kuijie Ankang Decoction on the expression level of p-AKT protein in colon tissue of UC model ratsAccording to gel electrophoresis imaging,PI3 KmRNA imaging density in colon tissues of rats in the model group was the highest,and its expression level was significantly higher than that in the normal group.After Kuijieankang Decoction and SASP intervention,PI3 KmRNA imaging density in colon tissues of rats in the above two groups decreased,and its expression level was lower than that in the model group.By the same method,according to gel electrophoresis imaging,the imaging density of AKTmRNA in the colon tissues of rats in the model group was the highest,and the expression level was significantly higher than that in the normal group.After the intervention of Kuitiangang Decoction and SASP,the imaging density of AKTmRNA in the colon tissues of rats in the above two groups decreased,and the expression level was lower than that in the model group.Compared with the control group,the protein expression of P-Akt in colon tissue of model group was significantly increased,and the difference was statistically significant(P<0.01).Compared with model group,the expression of P-Akt protein was significantly decreased in treatment groups after intervention,with statistically significant difference(P<0.01).There was no significant difference in the expression of P-Akt in colon tissue between Kuijie Ankang Decoction group and SASP group(P > 0.05).Conclusion:1.Kuijiankang Decoction can effectively relieve the general state(including mental state,diet state,defecation,blood in stool,body mass,etc.)and colonic morphological changes(effectively reduce CMDI score)of UC model rats,as well as reduce the histopathological changes of inflammatory cell infiltration in colonic tissue of UC model rats and change the intestinal ring of UC rats through HE staining It can reduce intestinal inflammation in UC model rats,which objectively proves that Kuijie Ankang Decoction can effectively improve intestinal inflammation and better protect damaged intestinal mucosal tissue in UC model rats,so as to achieve the purpose of treatment.2.Kuijie Ankang Decoction can maintain the balance of intestinal mucosal immune system by reducing the concentration of NO in the serum of UC model rats,inhibit inflammatory response,reduce cell damage,protect intestinal mucosa and promote ulcer healing.3.Kuijie Ankang Decoction can reduce the expression of VEGF mRNA in the colon tissue of UC rats,improve the local intestinal hypoxia environment,inhibit pathological vascular hyperplasia,reduce intestinal mucosal cell atrophy,and then promote the healing of mucosal ulcer.4.Kuijie Ankang Decoction can down-regulate the expression levels of PI3 K mRNA and AKT mRNA and down-regulate the expression level of p-AKT protein in colon tissue of UC model ratsThe increased expression of p-AKT is often regarded as a marker for the activation of PI3K/AKT signaling pathway,and phosphorylation is a necessary condition for the activation of AKT.Therefore,Kuijie Ankang Decoction can down-regulate the expression level of p-AKT protein in UC model rats.Effectively inhibit the PI3K/AKT signaling pathway,further block the conduction of the downstream pathway,and then control the release of biochemical mediators such as NO and VEGF and pro-inflammatory factors caused by the downstream pathway,thus achieving the purpose of treating UC.In summary,this experimental study concluded that Kuijieankang Decoction could effectively inhibit the PI3K/AKT signaling pathway,down-regulate the concentration of NO in serum of UC model rats,and reduce the expression of VEGF gene in colonic tissue of model rats.It was further confirmed that Kuijieankang Decoction reduced the serum NO in UC model rats.The concentration of NO and the expression of VEGF gene may be related to the mediation of PI3K/AKT signaling pathway.