Ginsenoside Derivative AD-1 Improves TNBS-Induced Acute IBD In Mice By Inhibiting The Activation Of NLRP3 Inflammasome

Background: As a traditional Chinese herbal medicine,ginseng has been widely used for health care and the treatment of certain chronic diseases.Ginsenoside is one of the main active components of ginseng,which has good anti-inflammatory,anti-tumor and other pharmacological effects.AD-1[20(R)-25-methoxy-dammarane-3β,12β,20-triol] is a new ginsenoside derivative.Inflammatory bowel disease(IBD)is a chronic intestinal inflammat-ory disease with unclear pathogenesis,recurrent episodes and severe complications.Therefore,finding new therapeutic methods is of great significance for improving the prognosis of IBD.NOD-like protein receptor 3(NLRP3)plays an important role in the development of IBD,but the specific role of AD-1 in the development of IBD is still unclear.Objective: To explore whether AD-1 has a therapeutic effect on TNBS-induced acute IBD mouse model,and whether the mechanism of action is to inhibit the activation of NLRP3 inflammasome to improve the degree of inflammation of the disease.Methods: Male C57BL/6 mice aged 6-8 weeks were randomly divided into normal control group(Control),ethanol control group(Alcohol),model group(TNBS),treatment group(AD-1)and positive group after 7 days of adaptive feeding.Control group(5-ASA),10 mice in each group,the latter three groups were pre-sensitized with 1% TNBS ethanol solution smeared on the back skin of mice every day 7 days before modeling,and the modeling day was fasted on 12 Hours later,2.5% TNBS ethanol solution was used for enema administration,and each mouse was injected with 100 μl.The normal control group and the ethanol control group were injected with the same volume of normal saline and25% ethanol solution by the same method as above.AD-1(10mg/kg)and positive control drug 5-ASA(30mg/kg)were respectively dissolved in 0.5% sodium carboxymethyl cellulose(CMC),and administered by gavage on the second day after enema modeling,the normal control group and the ethanol control group were respectively gavaged with CMC,200 μl per mouse,for 4 consecutive days,and the mice were sacrificed on the 6th day.The disease activity index(DAI)of the mice was monitored throughout the experiment;after the modeling,the colons of the mice were taken and the length of the colon was measured;HE staining was used to observe the pathological changes of colon inflammation in the mice;RT-PCR and WB experimental methods were used The expression levels of pro-inflammat-ory cytokines,claudin and NLRP3 inflammasome in colon tissue of mice were detected;the expression levels of serum IL-1β and IL-18 were detected by ELISA.Data were statistically processed using Graphpad prism7 software.Results:1.AD-1 significantly improves body weight loss in TNBS-induced acute IBD mice,reduces DAI score and alleviates colonic length shortening2.HE staining results showed that in the model group,the destruction of crypt structure increased,the goblet cells decreased,and a large number of inflammatory cells infiltrated.3.RT-PCR method was used to detect the expression of pro-inflammatory cytokines in colon tissue.The results showed that compared with the model group,the m RNA expressions of IL-6(P < 0.01)and TNF-α(P < 0.05)in the colon tissue of the treatment group were higher than those of the model group.decrease,statistically significan.4.RT-PCR method was used to detect the expression of NLRP3 inflammasome and downstream cytokines in colon tissue.The results showed that compared with the model group,NLRP3(P < 0.01),ASC(P < 0.001),Caspase-1 and Caspase-1 were significantly higher in the colon tissue of the treatment group than the model group.(P < 0.01),IL-1β(P< 0.001),and IL-18(P < 0.01)m RNA expressions were all decreased,with statistical significance.5.WB method was used to detect the expression of pro-inflammatory cytokines in colon tissue of mice.The results showed that compared with the model group,the protein expressions of IL-6(P < 0.01)and TNF-α(P < 0.05)in the colon tissue of the treatment group were higher than those of the model group.decreased,with statistical significance.6.WB method was used to detect the expression of NLRP3 inflammasome and downstream cytokines in colon tissue.The results showed that compared with the model group,NLRP3(P < 0.001),ASC(P < 0.01),and Caspase-1(P < 0.001)were significantly higher in the treatment group than in the model group.IL-1β(P < 0.05),and IL-18(P <0.01)protein expression levels were significantly decreased,with statistical significance.7.Western blot immunoblotting method was used to detect the expression levels of tight junction proteins ZO-1 and Occludin in colon tissue.The results showed that compared with the model group,ZO-1(P <0.01)and Occludin(P <0.001)The expression increased,which was statistically significant.8.The expression of IL-1β and IL-18 protein in serum of mice was detected by ELISA,and it was found that AD-1 significantly decreased the contents of IL-18 and IL-1β(P <0.05),which was statistically significant.Conclusion: The ginsenoside derivative AD-1 has the effect of improving TNBS-induced acute IBD in mice,and its mechanism may be to reduce the degree of inflammation by inhibiting the activation of NLRP3 inflammasome.