| Background and objecyive:Pioglitazone is a ligand of peroxisome proliferator activated receptor gamma (PPAR-γ). Recently, researches showed that after ligand activation, PPAR-γ, a potential target in the treatment of inflammation bowel disease(IBD), can reduce the transcription and expression of proinflammatory factor through inhibition of NF-κB activation, thus down-regulate excessive immune inflammatory reaction. The activation of TLRs signaling pathway can activate NF-κB, thereby induce the production of proinflammatory factor, which plays an important role in the pathogenesis of IBD. While we know rarely about the interrelationship between anti-inflammatory PPAR-γsignaling pathway and pro-inflammatory TLRs signaling pathway. In our study, we observed the therapeutic effect of pioglitazone on DSS-induced experimental colitis, as well as the expression of PPAR-γ, the key factors of TLRs signaling pathway- TLR2, TLR4, MyD88, the negative regulatory factor of TLRs signaling pathway-SIGIRR and the active marker of NF-κB- NF-κB p65, thus to study the relationship between PPAR-γsignaling pathway and TLRs signaling pathway in IBD and discuss the mechanism of the effect of pioglitazone on IBD from a new viewpoint.Method:(1)Groups:①Control group: only given drug vehicle-0.5% CMC;②Model group: only given drug vehicle-0.5% CMC;③SASP group: given 100mg/kg SASP+0.5%CMC;④The high dose group of PG(PG1): given 100mg/kg PG+0.5%CMC;⑤The mean dose group of PG(PG2): 50mg/kg PG+0.5%CMC;⑥The low dose group of PG(PG3): 25mg/kg PG+0.5%CMC. 10 mice each group.(2)Establishment of DSS-induced colitis and intervention of drugs:BALB/c mice were given 3% DSS ad libitum for 7 days, then distilled water for another 10 days. Chronic colitis model was successfully established according to anyone of the symptoms, loose stools, diarrhea, positive occult blood and bloody stools. Mice given distilled water only were used as the control group. After that, mice were randomly divided into groups mentioned above and drugs were given by intragastric administration once a day, from the second day of model building to the end. Then mice were sacrificed and samples were collected. Totally 18 days.(3) Observation and detection of each index:①To observe the weight and disease activity index (DAI) of each group everyday;②To observe the magnum shape of colon in mice macroscopically, and the change of pathohistology through HE stain;③To detect the expression of TLR2, TLR4, MyD88, SIGIRR and PPAR-γmRNA in colon through RT-PCR;④To detect the protein expression of TLR2, TLR4, MyD88 and NF-κB p65 through immunohistochemistry stain.Result:(1)Weight and DAI score of mice①Except the control group, the weights of mice in each group were significantly declining on the 5th day, then rose gradually on the 10th day and the 15th day. The weight descending percentages of model group and drug groups were higher than that of control group on the 5th, 10th, 15th day(P<0.001, 0.005, 0.05);the weight descending percentages of SASP group and PG1,PG3 group were lower than that of model group on the 5th day(P<0.05). On the 10th day, the weight descending percentage of PG3 group was lower than that of model group and SASP group(P<0.05). On the 15th day ,the weight descending percentage of SASP group was lower than that of model group and PG2 group(P<0.05),the weight descending percentage of PG1 group was lower than those of model group and PG2, PG3 groups(P<0.05,0.005,0.001).②There were significant deviation of DAI scores of each group on the 5th, 10th day, the DAI scores of control group were significantly lower than those of model group and each drug group(P<0.001); on the 5th day the scores of SASP group and PG1 group were significantly lower than those of model group(P<0.001, 0.05), the scores of PG1 group were significantly lower than those of PG2 group(P<0.05); on the 10th day the scores of PG3 group were significantly lower than those of model group, SASP group and PG2 group(P<0.05). There was deviation during DAI scores of each group, but without statistical significance on the 15th day.(2)Pathological change of colon①Magnum shape of colon mucosa: There was no congestion and edema in colon mucosa of control group; there was obviously congestion and edema in colon mucosa of model group; the symptoms of congestion and edema of SASP group and PG groups were lighter than those of model group in colon mucosa; visible ulcer and erosion can not be seen on colon of each group through macroscopic observation.②The pathohistological severity score of colon: The degrees of colon inflammation, pathological depth and crypt destruction in control group were significantly lower than those in other groups(P<0.001, 0.005);the degrees of those indexes in SASP group,PG1 and PG2 group were significantly lower than those in model group(P<0.001, 0.005, 0.05); the degree of colon inflammation in PG1 group was significantly lower than that in PG3 group(P<0.05).(3)TLR2 expression in colon mucosa: There was no significant deviation in the expression of TLR2 mRNA in colon mucosa of each group; but, the protein expression of TLR2 of control group was significantly lower than that of model group and each drug group(P<0.001), the expressions of SASP group,PG1 group and PG2 group were significantly lower than those in model group(P<0.05), the decreased expressions of PG3, PG2, PG1 groups were dose-dependent, but without statistical significance(P>0.05).(4) TLR4 expression in colon mucosa: The expression of TLR4 mRNA in colon mucosa of model group was significantly higher than that of control group(P<0.001, 0.01),the decreased expressions of PG3, PG2, PG1 groups were dose-dependent, but without statistical significance(P>0.05), and there was no significant deviation between drug groups and control group(P>0.05). The protein expression of TLR4 in colon mucosa of control group was significantly lower than that of model group and drug groups(P<0.001, 0.005),the expressions of SASP group,PG1 and PG2 groups were significantly lower than those of model group(P<0.01, 0.05),the decreased expressions of PG3, PG2, PG1 groups were dose-dependent, the expression of PG1 group was significantly lower than that of PG3 group(P<0.05).(5) MyD88 expression in colon mucosa: The expression of MyD88 mRNA in colon mucosa of model group was significantly higher than that of control group,SASP group and PG1 group(P<0.001, 0.05), the decreased expressions of PG3, PG2, PG1 groups were dose-dependent, but without statistical significance(P>0.05), and there was no significant deviation between drug groups and control group(P>0.05). The protein expression of MyD88 in colon mucosa of control group was significantly lower than that of model group and drug groups(P<0.001) , the expressions of SASP group,PG1 and PG2 group were significantly lower than that of model group(P<0.05) , the decreased expressions of PG3, PG2, PG1 groups were dose-dependent, but without statistical significance(P>0.05).(6)SIGIRR mRNA expression in colon mucosa: The expression of SIGIRR mRNA in colon mucosa of control group was significantly higher than that of model group, SASP group, PG2 and PG3 group(P<0.005, 0.01, 0.05),there was no significant deviation between drug groups and model group(P>0.05),the increased expressions of PG3, PG2, PG1 groups were dose-dependent, but without statistical significance(P>0.05).(7) PPAR-γmRNA expression in colon mucosa: The expression of PPAR-γmRNA in colon mucosa of control group was significantly higher than that of model group,PG2 and PG3 group(P<0.005, 0.01, 0.05),the expression of SASP group was significantly higher than that of model group(P<0.05),the increased expressions of PG3, PG2, PG1 groups were dose-dependent, but without statistical significance(P>0.05).(8) NF-κB p65 protein expression in colon mucosa: The protein expression of NF-κB p65 in colon mucosa of control group was significantly lower than that of model group and drug groups(P<0.001,0.005,0.01), the expressions of SASP group and PG1 group were lower than those of model group(P<0. 05),the decreased expressions of PG3, PG2, PG1 groups were dose-dependent, but without statistical significance(P>0.05).Conclusion:(1)The expression of PPAR-γand SIGIRR was decreased in the colon mucosa of DSS-induced experimental colitis mice, while the expressions of TLR2, TLR4, MyD88, NF-κB p65 increased, which indicated that the over-activation of TLRs/NF-κB signaling pathway and the decrease of its negative regulatory mechanism, as well as the inhibition of PPAR-γsignaling pathway participate in the pathopoiesis of IBD.(2)Pioglitazone, the ligand of PPAR-γ, can ease the symptoms of colon and attenuate the inflammation and injury of pathohistology, which can be taken as a new approach in the treatment of IBD.(3)The expressions of TLR2, TLR4, MyD88, NF-κB p65 significantly decreased in colon mucosa of mice which were given pioglitazone, and were concerned with the attenuation of colon inflammation, suggesting that PPAR-γligand may attenuate the tissue inflammation through the inhibition of TLRs/NF-κB activation.(4)The expressions of SIGIRR and PPAR-γup-regulated dose-dependently in colon mucosa of mice which were given pioglitazone, suggesting that PPAR-γligangd may regulate the TLRs/NF-κB signaling pathway negatively through SIGIRR and PPAR-γ.
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