Qingchang Huashi Recipe Attenuates Ulcerative Colitis By Mechanisms Involving Regulation Of IL-6/STAT3/SOCS3 Signaling

Objective Targeting the Interleukin-6/Janus kinase 2/Signal transducer and activator of transcription 3/Suppressor of cytokine signaling 3 pathway,this paper aims to explore the possible treatment mechanism of Qingchang Huashi Recipe(QHR) in vitro using HT-29 cell line stimulated by hTNF-a and LPS and in vivo using experimental colitis mice induced by trinitrobenzene-sulfonic acid/ethanol.Methods In vitro,The HT-29 cell was stimulated by hTNF-a and LPS.HT-29 cells were divided into 6 groups,as followed:the model group,the SASP group,the high dose QHR group, the medium dose QHR group, the low dose QHR group. The concentration of IL-6 was detected by ELISA,The expression of JAK2、STAT3 and SOCS3mRNA were detected by Real Time PCR. The expression of p-JAK2、p-STAT3 and SOCS3 protein were detected by Western Blot. In vivo, the colitis model of mice was established with TNBS/ethanol.Forty mice were randomly divided into four groups (n=10):control group, model group,QHR group, and SASP group.After continuous administration for 7 d,the colon tissue level of sIL-6R were detected using EL1SA, the mRNA expression of IL-6、gp130、JAK2、STAT3 and SOCS3 in the colon tissues of mice were detected using Real Time PCR,and the protein expression of IL-6 gp130、p-JAK2、p-STAT3 and SOCS3 in the colon tissues of mice were detected using Western Blot.Results In vitro, the secretion of IL-6, the protein expression of p-JAK2 and p-STAT3, the expression of SOCS3 mRNA and protein in HT-29 were significantly higher in model group than in control group (all P< 0.01). The secretion of IL-6, the expression of p-JAK2、p-STAT3 protein in all QHR groups significantly reduced compared to the model group in a dose-dependent manner (all P< 0.01).Elevated SOCS3 mRNA and protein expression was observed in the high dose QHR group and the medium dose QHR group compared to the model group (P＜0.01).No significant difference of JAK.2 and STAT3 mRNA expression was detected. (P<0.05).In vivo, QHR could lower both the pathology score and the general morphology score of colon. The level of sIL-6R, the gene and protein expression of IL-6、gp130、SOCS3, the protein expression of p-JAK2 and p-STAT3 in colon tissues were significantly higher in model group than those in control group (all P< 0.01). QHR significantly reduced the sIL-6R level (P< 0.01), the mRNA and protein expression of IL-6 and gp130, and the the protein expression of p-JAK2 and p-STAT3 in the colon tissues (all P＜0.01). Elevated SOCS3 mRNA and protein expression was detected in all QHR groups compared to the model group (P<0.01). No significant difference of JAK2 and STAT3 mRNA expression was detected(P<0.05).Conclusion The results of experiment in vitro showed that QHR might influence hTNF-a and LPS stimulated HT-29 cells by up-regulating SOCS3,suppressing JAK2 and STAT3 activation,decreasing IL-6. The results of experiment in vivo showed that QHR exibited anti-inflammatory effect on experimental colitis which might involve up-regulation of SOCS3, inhibition of JAK2 and STAT3 activation, suppression of IL-6 trans-signaling.