| Background:IOP(Inonotus Obliquus Polysaccharide,IOP)is a polysaccharide with various biological activities extracted from Inonotus obliquus.Current research shows IOP has biological effects against a variety of tumors.CAC(Colitis-associated colorectal cancer,CAC)is a common type of colorectal tumor.At present,from the multidisciplinary and multiangle get a large amount of supporting evidence to prove that colon inflammation is one of the main causes of colorectal tumors.NLRP3 is shown that the development is closely related to CAC,but the specific role in the development of CAC is still unclear.Objective:To clarify the impact of IOP on CAC model mice,and to explore whether its mechanism of action is related to NLRP3 inflammatory bodies.Methods:After 7 days of adaptive feeding of six to eight week-old Balb/C male mice,mice were randomly divided into three groups.The CAC animal model was constructed by treatment with AOM and DSS.The first group is the normal control group.10 days in advance,with 0.9%sterile saline 0.3 ml/only,the next day.After 10 days,a single intraperitoneal injection of the same volume of sterile saline as the second group of AOM,fresh drinking water is provided until the end of the experiment.The second group is the AOM/DSS+saline model group,referred to as the model group.10 days in advance,with 0.9%sterile saline 0.3 mL/only every other day.After 10 days,mice were given 10 mg/kg body weight of AOM by intraperitoneal injection.The mice were then given 2.5%DSS water for 7 days,followed by 14 days of normal water.This cycle was repeated four.The third group is the AOM/DSS+IOP experimental group,referred to as the IOP group.10 days in advance,150 mg/kg/0.3 mL IOP solution was administered intragastrically every other day until the end of the experiment.The AOM/DSS modeling scheme is the same as the second group.All mice were sacrificed by sacrifice on day 102.During the experiment,every 7 days as the nodes,and the rate of change in body weight was calculated and analyzed,and record the number of mice surviving during the experiment.The colon tissue of the mice was dissected and the length of the colon,the number of tumors,the longer diameter of the tumor,and the tumor load were measured.Mouse colon tissue was treated by HE staining and observed under a microscope.Treatment of mouse colon tissue by IHC staining,the expression of NLRP3,Caspase-1 protein,cand proinflammatory cytokines IL-18,IL-1β,COX-2 were observed.The mRNA levels of NLRP3,Caspase-1,ASC,IL-18,IL-1β,IL-6 and TNF-α in mouse colon tissues were detected by RT-PCR.The protein levels of NLRP3,ASC,Caspase-1,IL-18,IL-1β,IL-6,TNF-a and COX-2 in mouse colon tissues were detected by Western Blot Data were statistically analyzed using GraphPad Prism 7 software.Results:1.IOP affects AOM/DSS-induced changes in body weight of model mice.On the 45th day,the body weight change rate of the IOP group was higher than that of the model group(P<0.05);On the 66th day,the body weight change rate of the IOP group was higher than that of the model group(P<0.05).In addition,from day 38 to day 101,the rate of change in body weigh6 in the IOP group was always higher than in the model group:2.IOP’ affects AOM/DSS-induced survival in model mice.By statistics,the survival rate of the model group was 41.67%,and the survival rate of the IOP group was 66.67%,which was significantly higher than that of the model group(P<0.01).3.IOP affects the occurrence of adenomas in AOM/DSS-induced model mice.The average number of adenomas in the model group was 24±4,the mean volume was 3.48 ±0.68 mm3,and the average tumor burden was 59.09±4.13 mm.The mean adenoma number in the IOP group was 14±3(P<0.01),the mean volume was 1.50 ± 0.46 mm3(P<0.01),and the mean tumor burden was 25.00±2.64 mm(P<0.01).4.HE pathological results showed that in the model group,the colonic epithelial cells were disordered,the mucous layer disappeared,there was no crypt structure,no goblet cells,a large number of inflammatory cells infiltrated,the gland structure had a sieve-like structure and back-to-back phenomenon and gland thickening,the ratio of nucleoplasmic pulp is increased,the nuclear staining is deep.In the IOP group,the intestinal mucosa structure was relatively complete,the mucus layer was localized,and the crypt structure disappeared locally,a small number of goblet cells,less inflammatory cell infiltration.5.IHC staining results show that compared with the model group,the expression of NLRP3,Caspase-1,IL-18 and IL-1β was increased in the IOP group,and the expression of the inflammation-related cytokine COX-2 was decreased.6.RT-PCR method to detect the expression of NLRP3 polyprotein complex and related cytokines in colon tissue,the results showed that compared with the colon tissue of the model group,NLRP3(P<0.01),ASC(P<0.05),Caspase-1(P<0.01),IL-18(P<0.01).In the IOP group,the expression of IL-1β(P<0.05)mRNA was elevated and statistically significant.The mRNA expressions of inflammatory cytokines IL-6(P<0.01)and TNF-a(P<0.01)were significantly decreased and statistically significant.7.WB method detects the expression of NLRP3 polyprotein complex and related cytokines in colon tissue,the results show,the expression levels of NLRP3(P<0.01),ASC(P<0.01),Caspase-1(P<0.01),IL-18(P<0.01),and IL-1β(P<0.05)were significantly higher in the IOP group.In the model group,the expression levels of pro-inflammatory cytokines IL-6(P<0.01),TNF-a(P<0.01),and COX-2(P<0.01)were significantly lower than those in the model group.Conclusion:IOP has a preventive effect on AOM/DSS-induced CAC model mice,and its mechanism may be related to activation of inflammatory steroid NLRP3 and reduction of its associated inflammatory cytokines. |
