Study On The Mechanism Of Qingchangshuan Decoction In Promoting Colonic Mucosal Repair Of Ulcerative Colitis Through IGF-1/β-arrestin 2/ERK Signaling Pathway

Objectives:1.To study the effect and mechanism of Qingchangshuan(QCS)decoction on mucoal repair in ulcerative colitis(UC)model rats induced by dextran sulfate sodium(DSS).2.To investigate the effect of QCS decoction-containing serum on DSS-induced Caco-2 cells injury model and its potential mechanism.Methods:1.In vivo: According to body weight in a completely randomized block design,40Sprague-Dawley male rats were divided into Normal group,Normal+QCS group,Model group,Model+QCS group and Model+sulfasalazine(SASP)group.The general conditions of rats in each group were observed and disease activity index(DAI),colon length,colon macroscopic damage index(CMDI)and histopathological score of colitis were also recorded.The levels of serum pro-inflammatory cytokines TNF-α IL-1β,IL-6and anti-inflammatory cytokine IL-10 in rats were measured by ELISA.The numbers of colonic epithelial cells and goblet cells in each group were counted by HE staining and AB-PAS staining.Immunohistochemistry and TUNEL were used to detect the expressions of Ki67 proliferation and apoptosis in colonic epithelial cells,respectively.The expressions of PCNA and Cleaved Caspase-3 were analyzed by Western-blot.The serum levels of IGF-1 in rats were detected by ELISA,and the related protein expressions of IGF-1/β-arrestin 2/ERK signaling pathway in colon tissue were determined by Western-blot.2.In vitro: Logarithmic growth phase Caco-2 cells were collected and divided into five groups: Normal group,Normal+QCS group,Model group,Model+QCS group and Model+QCS+IGF-1R inhibitor group.Cell viability was detected by CCK assay.Cell migration ability in vitro was tested by scratch test.The contents of TNF-α,IL-1β,IL-6and IL-10 in the supernatant of cultured cells were determined by ELISA.The expressions of PCNA and Cleaved Caspase-3 were detected by Western-blot.The related protein expressions of IGF-1/β-arrestin 2/ERK signaling pathway were also determined by Western-blot.Results:1.In vivo:(1)The rats in the Model group looked sluggish and suffered from weight loss,loose and bloody stool.Compared with the Normal group,the Model group had a higher DAI score,a shortened length of the colon,an significant increase in CMDI and a higher pathological score of colitis.The levels of serum pro-inflammatory cytokines TNF-α,IL-1β,IL-6 increased while the contents of anti-inflammatory cytokine IL-10 decreased.QCS decoction and SASP could effectively improve the general conditions,the changes of the aboved parameters and inflammatory situation of UC model rats induced by DSS,but there is no significant difference between Model+QCS group and Model+SASP group.(2)QCS decoction could restore the declining numbers of colonic epithelial cells and goblet cells in UC model rats induced by DSS.It also effectively reduced apoptosis of colon epithelial cells and the expression of Cleaved Caspase-3 while up-regulated the proliferation of Ki67 in colon epithelial cells and the protein expression of PCNA.(3)QCS decoction could increase the contents of serum IGF-1 in DSS-induced model rats,up-regulated the protein expressions of p-IGF-1R,β-arrestin 2,p-ERK1/2 protein in the IGF-1/β-arrestin 2/ERK signaling pathway,and increased the ratio of p-IGF-1R/IGF-1R and p-ERK1/2/ERK/1/2 significantly.2.In vitro: QCS decoction-containing serum could antagonize the decrease of Caco-2cell viability induced by DSS,accelerate the healing of scratches and promote cell migration activity in vitro.It also significantly reduced the secretion of TNF-α,IL-1β,IL-6 induced by DSS while increased the release of IL-10.It promoted the activation of IGF-1/β-arrestin 2/ERK signaling pathway.Addition of IGF-1R inhibitors delayed the recovery of cell injuries and the healing speed of scratches.The cell migration in vitro was also inhibited.The level of TNF-α was slightly rised and the content of IL-10 was reduced,however,the IL-1β and IL-6 levels were unchanged.It also inhibited the increase of PCNA levels and promoted the expression of Cleaved Caspase-3correspondingly.Meanwhile,the related protein expressions of IGF-1/ β-arrestin 2/ERK signaling pathway were down-regulated after adding inhibitor of IGF-1R.Conclusions:1.QCS decoction could effectively improve the colon injury induced by DSS in UC model rats.It inhibited inflammation and cell apoptosis as well as promoted the proliferation of colonic epithelial cells via IGF-1/β-arrestin 2/ERK signaling pathway so as to accelerate the repair of colonic mucosa.2.QCS decoction-containing serum could protect Caco-2 cells from DSS-induced injury.It could improve cell survival rate through inhibiting proinflammatory cytokines,promoting cell migration and activating the IGF-1/β-arrestin 2/ERK signaling pathway of cell proliferation.