| Objective1.To observe the regulation of herbal cake-partitioned moxibustion on inflammatory factors,inflammatory pathway and autophagy-related indicators in CD rats.2.To clarify that the herbal cake-partitioned moxibustion inhibits the excessive activation of autophagy in the colon tissue of CD rats by regulating the PI3KC1-related signaling pathway.Provide scientific basis for herbal cake-partitioned moxibustion treatment of CD.3.To observe the effect of herbal cake-partitioned moxibustion on mi RNAs in serum exosomes of CD rats,and to verify the differential mi RNAs related to autophagy,further explore the overall effect of herbal cake-partitioned moxibustion on CD.Methods1.Male SD rats were randomly divided into normal group,model group and herbal cake-partitioned moxibustion group.Preparation of rat Crohn’s disease model by 2,4,6-trinitrobenzenesulfonic acid(TNBS)enema.On the basis of the model group,the rats in the herbal cake-partitioned moxibustion group were given herbal cake-partitioned moxibustion on Qihai acupoint and both sides of Tian Shu acupiont for treatment.Identification model by by gross visual observation,macroscopic observation of colon,and pathological changes of colon tissue.Relevant indicators were detected by RT-q PCR,WB and Immunostaining.The changes of inflammatory factors(IL-18,TNF-a),inflammatory pathway(TLR4/My D88/NF-k B)and autophagy-related indicators(LC3B,p62,Beclin-1,p-m TOR)in rats colon tissue were observed.2.Male SD rats were randomly divided into model group,insulin+model group,insulin+herbal cake-partitioned moxibustion group,rapamycin+model group,rapamycin+herbal cake-partitioned moxibustion group.Rats in each group were treated with 2,4,6-trinitrobenzenesulfonic acid(TNBS)enema to prepare Crohn’s disease model.Administration of insulin and rapamycin by intraperitoneal injection.Rats in each group were observed with gross visual observation,macroscopic observation of colon,and pathological changes of colon tissue.Relevant indicators were detected by RT-q PCR,WB.The changes of PI3 KCs signaling pathway related indicators and autophagy related indicators(PI3KC1,Akt,m TOR,PI3KC3,LC3 B,p62)in rats colon tissue were observed.3.Differentially expressed mi RNAs in serum exosomes of each group(From experiment 1)were screened by Illumina Hi Seq TM 2500 sequencing and target gene prediction was performed.Verification of selected mi RNAs by using RT-q PCR and luciferase reporter.Results Section one 1.Model identification: 1.Model identification: After modeling,compared with the normal group rats,the hair of the model group rats was yellow,rough and messy,the food intake was poor,the perianal was stained with loose stools,the litter was messy,and the smell was strong.After exposing the abdominal cavity,it was observed that the colon was significantly shortened and thickened,and there was obvious gas accumulation in the intestine,and intestinal adhesions,megacolon or intestinal obstruction were observed in some rats.After cutting the intestine,it was observed that the surface of the intestinal mucosa was not smooth,the intestinal wall was thickened,accompanied by scattered edema,congestion,ulcers,etc.,and the surface of the ulcer was stained with feces and was not easy to rinse and some rats showed visible paving stone changes,suggesting that the model was successfully prepared.2.Effect of herbal cake-partitioned moxibustion on m RNAs of inflammatory factors and pathway indicators in colon tissue of CD rats: Compared with the normal group,the expression levels of IL-18,TNF-α,TLR4,My D88,and NF-k B p65 m RNA in the colon tissue of the model group were significantly increased(P<0.01).Compared with the model group,the expression levels of IL-18,TNF-α,TLR4,My D88,and NF-k B p65 m RNA in the herbal cake-partitioned moxibustion group were significantly decreased(P<0.05).3.Effect of herbal cake-partitioned moxibustion on proteins of inflammatory factors and pathway indicators in colon tissue of CD rats: Compared with the normal group,the expression levels of IL-18,TLR4,My D88 and NF-k B p65 proteins in the colon tissues of the model group were significantly increased(P<0.01).Compared with the model group,the expression levels of IL-18,TLR4,My D88,and NF-k B p65 proteins in the herbal cake-partitioned moxibustion group were significantly decreased(P<0.05).Compared with the normal group,the expression level of TNF-α protein in colon tissue of the model group was increased,however,the difference was not statistically significant(P=0.116).Compared with the model group,the expression level of TNF-α protein in colon tissue of the herbal cake-partitioned moxibustion group was decreased,but the difference was not statistically significant(P=0.575).4.Effect of herbal cake-partitioned moxibustion on the autophagy related indicators of colon tissue in CD rats: Compared with the normal group,the m RNA and protein expression levels of LC3 B,Beclin-1 in colon tissue of rats in the model group were increased significantly(P<0.01).Compared with the model group,the m RNA and protein expression levels of LC3 B,Beclin-1 in colon tissue of rats in the herbal cake-partitioned moxibustion group were were decreased significantly(P<0.05).Compared with the normal group,the m RNA and protein expression levels of m TOR,p62 in colon tissue of rats in the model group were decreased significantly(P<0.01).Compared with the model group,the m RNA and protein expression levels of m TOR,p62 in colon tissue of rats in the herbal cake-partitioned moxibustion group were increased significantly(P<0.05).Section Two 1.Effect of herbal cake-partitioned moxibustion on m RNAs of PI3KC1 signal pathway and autophagy indicators in colon tissues of CD rats: Compared with the model group,the expression levels of PI3KC1,Akt1,m TOR and p62 m RNAs in the colon tissue of the rats in the rapamycin+model group were down-regulated in different degrees.Compared with the rapamycin+model group,the expression levels of PI3KC1,Akt1,m TOR and P62 m RNAs in the colon tissue of the rats in the rapamycin+herbal cakepartitioned moxibustion group were up-regulated significantly(P<0.05).Compared with the model group,the expression level of LC3 B m RNA in the colon tissue of the rats in the rapamycin+model group was up-regulated.Compared with the rapamycin+model group,the expression level of LC3 B m RNA in the colon tissue of the rats in the rapamycin+herbal cake-partitioned moxibustion group was down-regulated significantly(P<0.05)2.Effect of herbal cake-partitioned moxibustion on proteins of PI3KC1 signal pathway and autophagy indicators in colon tissues of CD rats: Compared with model group,the expression levels of PI3KC1,Akt1,p-m TOR and p62 proteins in the colon tissue of the rats in the rapamycin+model group were down-regulated in different degrees.Compared with rapamycin+model group,the expression levels of PI3KC1,p-Akt,pm TOR,LC3 B and p62 proteins in the colon tissue of the rats in the rapamycin+herbal cake-partitioned moxibustion group were up-regulated significantly(P<0.05).Compared with the model group,the expression levels of VPS34(PI3KC3),LC3 B protein in the colon tissue of the rats in the rapamycin+model group were up-regulated.Compared with rapamycin+model group,the expression levels of VPS34(PI3KC3),LC3 B protein in the colon tissue of the rats in the rapamycin+herbal cake-partitioned moxibustion group were down-regulated significantly(P < 0.05).Section Three 1.High-throughput sequencing of mi RNAs in rats serum exosomes: There were 26 mi RNAs with extremely significant differences(P<0.01)between the normal group and the model group,and 41 mi RNAs with significant differences(P<0.05).There were 15 mi RNAs with extremely significant differences(P<0.01)between the model group and the herbal cake-partitioned Moxibustion group,and 27 mi RNAs with significant differences(P<0.05).Most of the differential mi RNAs in the model group were upregulated compared with the normal group and most of the differential mi RNAs in the herbal cake-partitioned moxibustion group were down-regulated compared with the model group.KEGG analysis showed that the target gene function of differentially expressed micro RNAs in each group was highly enriched in PI3K-Akt and other signaling pathways.2.Screening and verification of autophagy-related mi RNAs: There are 7 extremely significant differential mi RNAs and 11 differentially significant mi RNAs between each group.By comparing the databases and reviewing the literature,the mi RNAs that are more likely to have an effect on autophagy are selected as rno-mi R-30b-5p,rno-mi R-142-3p,rno-mi R-106b-5p,rno-mi R-30c-5p.RT-q PCR verified that expression trends were consistent with sequencing trends.The luciferase reporter indicates that ATG12 and ATG5 are target genes of mi R-30b-5p.Conclusion 1.Herbal cake-partitioned moxibustion can inhibit the expression of related inflammatory pathway(TLR4/My D88/NF-k B)and inflammatory factors(IL-18,TNF-alpha),inhibit the hyperactivated autophagy in the process of inflammation,thereby alleviating the symptoms and pathological changes of TNBS-induced CD rats,and exerting therapeutic effects.2.Herbal cake-partitioned moxibustion may activating the PI3KC1/Akt/m TOR signaling pathway and inhibit the PI3KC3(VPS34)-associated protein complex,thus playing a role in inhibiting autophagy.3.Herbal cake-partitioned moxibustion can down-regulate the expression of most micro RNAs in serum exosomes of CD rats induced by TNBS.Among them,4 mi RNAs(rno-mi R-30b-5p,rno-mi R-142-3p,rno-mi R-106b-5p,rno-mi R-30c-5p)RT-q PCR results are consistent with sequencing results,and ATG12 and ATG5 were verified as target genes for mi R-30b-5p,suggested that the herbal cake-moxibustion may play a role in regulating autophagy by inhibiting mi R-30b-5p in serum exosomes of CD rats. |
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