| Objective:To investigate the effect of the specific inhibitor of Smad3(SIS3)on the inhibition of TGF-?1/Smad3 signaling pathway in rat intrauterine adhesion(IUA),and its relationship between transforming growth factor-?1(TGF-?1),connective tissue growth factor(CTGF)and matrix metalloproteinase-9(MMP-9).The correlation between the three factors was also analyzed to study the correlation between TGF-?1/Smad3 signaling pathway and the three factors.Methods:(1)The method of rat intrauterine adhesion modeling was selected:Eighteen healthy female SD rats aged 8-12 weeks were randomly divided into three groups.One group was modeled by intrauterine infusion of anhydrous ethanol,and the other group was modeled by mechanical injury combined with lipopolysaccharide infection,the control group(intrauterine injection of normal saline).Uterine tissues were collected 2 weeks after surgery,HE staining and Masson staining were used to detect the number of endometrial glands and the degree of endometrial fibrosis,and the semi-quantitative score of endometrial fibrosis was calculated,select the appropriate modeling scheme according to the results and enter into the subsequent study.(2)Grouping:After the modeling,thirty healthy female SD rats was randomly divided into a control group,a model group(an intrauterine adhesion model)and an intervention group(the SIS3 was given after the model was created).Uterine tissues were collected 2 weeks after surgery,HE staining and Masson staining were used to detect the number of endometrial glands and the degree of endometrial fibrosis,and the semi-quantitative score of endometrial fibrosis was calculated.The relative expression of TGF-?1,CTGF and MMP-9 mRNA and protein were detected by Reverse transcription PCR and Western-blot,respectively.the correlation analysis was also performed with the degree of endometrial fibrosis.The correlation analysis was also performed on the expression of the three factors.Results:(1)The scores of endometrial fibrosis in the control group,the anhydrous ethanol intrauterine infusion group and the mechanical injury+LPS infection group were 0,5.7±0.82 and 6.8±0.75,respectively.There were significant differences between groups(P<0.001).The intrauterine adhesion score in the anhydrous ethanol group and the mechanical injury+LPS infection group was higher than the control group(P<0.001).The score of mechanical injury+LPS infection group was higher than that of anhydrous ethanol group,but no statistics significance(P>0.05).However,the rats had severe adhesion in the abdominal cavity after modelling with anhydrous ethanol,and tight adhesion between the uterus and abdominal tissues,which was not conducive to the collection of subsequent experimental tissue samples.Therefore,mechanical injury+LPS infection method was used to establish the rat intrauterine adhesion models.(2)In subsequent experiments,from the control group,the intervention group to the model group,the number of endometrial glands gradually decreased,and the content of interstitial collagen fibers gradually increased.The scores of endometrial fibrosis in the three groups were 0,4.1±0.99,and 7.0±0.82,respectively,Compared with each other,the differences were statistically significant(P<0.001).(3)The expression levels of TGF-?1,CTGF and MMP-9 mRNA and protein in the model group were higher than those in the control group(P<0.05).The expression level of CTGF mRNA and protein in the model group were higher than those in the intervention group(P<0.05),but there was no significant difference in the expression levels of TGF-?1 and MMP-9 mRNA and protein in the two groups.The expression levels of TGF-?1 and CTGF mRNA and protein in the intervention group were higher than those in the control group(P<0.05).There was no significant difference in MMP-9 expression between the two groups.(4)The expression levels of CTGF mRNA and protein in the intervention group were positively correlated with endometrial fibrosis scores,but there was no significant correlation between TGF-?1 and MMP-9.(5)The expression levels of TGF-?1,CTGF mRNA and protein in the model group were positively correlated with the endometrial fibrosis score,while MMP-9 had no significant correlation.(6)There was no significant correlation between TGF-?1 and CTGFmRNA and protein in the intervention group,but was a positively correlated with the model group.No significant correlation was found between TGF-?1,CTGF and MMP-9 in mRNA and protein in both the intervention group and model group.Conclusions(1)Inhibition of TGF-?1/Smad3 pathway can reduce the degree of intrauterine adhesion after endometrial damage.(2)TGF-?1,CTGF and MMP-9 may involved in the pathogenesis of IUA.(3)There is a positive correlation between TGF-?1 and CTGF with the degree of intrauterine adhesion.(4)CTGF can be used as a potential molecular marker for intrauterine adhesion. |
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