| Objective: To observe the effect of Taohong Siwu Jiawei Decoction combined with adipose mesenchymal stem cells on the levels of inflammatory factors and fibrosis markers in rats with intrauterine adhesion,and explore its possible mechanism.Methods: A total of 60 healthy,non-pregnant female SD rats(10 to 12 weeks of age)were included,10 were selected as the sham operation group According to the method of random number table.The uterine adhesion model of rats was established by double damage method.Seven days after the establishment of the model,3 rats were randomly selected for morphological and pathological observation to verify the model.The rats randomly were divided into five groups:model group,crosslinked sodium hyaluronate gel group,traditional Chinese medicine group,ADSCs transplantation group and combination group,with 10 rats in the combination group and 9 rats in the remaining groups.In the sham operation group,abdominal cavity was opened,bilateral uterus was exposed,no uterus was treated,abdomen was closed,and then routine feeding was performed.Model group: After the completion of rat modeling,the same as sham operation group.The other rats were divided into groups for intervention and crosslinked sodium hyaluronate gel group: After laparotomy,0.5ml of self-crosslinked sodium hyaluronate gel was injected into the injured uterine tissue in situ.In Chinese medicine group,the dosage of Taohong Siwu jiawei doction solution 12g/Kg/d was given by gavage;In the ADSCs group,0.2ml ADSCs was transplanted into the injured uterine tissue after laparotomy.Combination group: After laparotomy,0.2ml ADSCs was transplanted into the uterine tissue of the injured segment,and at the same time,Taohong Siwu jiawei doction solution was administered intragastric administration,which was the same as the traditional Chinese medicine group.After 28-days of treatment,the gross morphology of uterus was observed by naked eye,and the morphological changes of uterus were observed by HE staining.Masson staining was used to analyze the fibrosis of endometrial tissue and calculate the fibrosis area.The expression levels of TNF-α,IL-6 and IL-18 in rat uterine tissue were detected by ELISA.The expressions of endometrial transforming growth factor(TGF-β1)and connective tissue growth factor(CTGF)were detected by immunohistochemistry.Results: 1.IUA model verification: After 7 days of modeling,3 model rats were randomly selected,and the gross anatomy showed that the surface of the uterus was red,the touch was poor,and the uterine wall was rough and irregular in shape.There were obvious blood clots,local edema,and effusion in the uterine cavity.HE staining showed that the uterine cavity was narrow,the uterine epithelium was discontinuous,fibrous adhesion was visible,there were lamellar flat epithelium on the surface of part of the uterine cavity,and there were a few glands in the basal layer,which was similar to oval shape,indicating successful modeling.2.General situation analysis of rats: All the rats in all groups were in good condition before the model was established,after the modeling,the rats in the model group had dull,sparse hair,cranky and irritable,increased secretions in the corner of the eye,bowed back,liked to curl up and huddle together,ate less,occasionally appeared blood in the urine,increased vaginal secretions,and unformed stool.After the intervention,the above conditions were improved to varying degrees in each treatment group.Self-crosslinking sodium hyaluronate group showed the most obvious improvement,followed by the combination group.3.Analysis of histopathologic changes of uterine tissue: Under the HE staining microscope,It was observed that the endometrium in the sham operation group was complete and uniform,covered with columnar epithelium,with abundant glands,which were open on the surface of the endometrium,mainly distributed in the submucosa and basal layer,and were oval or oblong.In the model group,inflammatory cells infiltrated the uterus,fibroblasts proliferated significantly,and other pathological manifestations were the same as those in the same group.In the other treatment groups,the shape of the uterus was irregular,the glandular epithelium and endometrial epithelium were basically intact,part of the endometrial surface was covered by single columnar epithelium,and different numbers of new glands could be seen under the epithelial layer,mostly round or oval.4.Analysis of endometrial fibrosis: 1)Qualitative analysis: a small amount of blue collagen fibers can be seen in the endometrial stroma of the sham operation group,arranged neatly and in the shape of spindles,and the mucosa,submucosa,blood vessels,and muscle layers are dark red;the model group The uterine cavity is narrow and almost completely closed.A large number of blue collagen fibers can be seen,arranged closely,forming a fibrous structure.Compared with the model group,each treatment group showed significant improvement,a small amount of fibrous adhesions appeared in some uterine cavities,which were in the shape of ropes,and the blue stained collagen fibers decreased to varying degrees.2)Semi-quantitative analysis: Compared with the sham operation group,the percentage of fiber area in the model group was significantly higher(P < 0.05);Compared with the model group,the proportion of fibrosis area in each treatment group was reduced to different degrees(P < 0.05),Crosslinked sodium hyaluronate group and combined group significantly decreased,but there was no statistical significance(P >0.05);The level of fibrosis in ADSCs was even greater when compared to the crosslinked sodium hyaluronate group(P<0.05);Compared with the combination group,there was no significant difference between the Taohong Siwu jiawei decoction group and the ADSCs transplantation group(P >0.05).5.Comparison of the contents of TNF-α,IL-6 and IL-18 In the uterus of rats: The concentrations of TNF-α,IL-6,IL-18 in the uterus of the model group were higher than those in the sham surgery group(P < 0.05);Compared with model group,IL-6,IL-18,and TNF-α were significantly reduced in all therapy groups,especially in cross-linked sodium hyaluronate and combined therapy,but the difference was not statistically significant(P >0.05);Compared with the crosslinked sodium hyaluronate group,the content of IL-6 in the Taohong Siwu jiawei decoction group was still higher(P < 0.05),and the contents of IL-18 and TNF-α were higher in ADSCs transplantation group(P < 0.05);Compared with the combination group,there was no significant difference between the Taohong Siwu jiawei decoction group and the ADSCs transplantation group(P >0.05).6.Comparison of the expression levels of TGF-β1 and CTGF in the uterine tissue of rats: compared with the sham operation group,TGF-β1 and CTGF expression in the uterus of model group were markedly elevated(P < 0.05);Compared with the model group,the expression levels of TGF-β1 and CTGF in the treatment groups were decreased to varying degrees,and the expression levels of TGF-β1 and CTGF in the crosslinked sodium hyaluronate group were the most significant,followed by the combination group,However,the difference was not statistically significant(P >0.05),while the expression level of TGF-β1 in Taohong Siwu jiawei decoction group,ADSCs transplantation group and combination group was slightly decreased,But no statistical significance was found(P >0.05),the expression level of CTGF in ADSCs transplantation group and combined group was slightly decreased,but there was no statistical significance(P >0.05);Compared with the crosslinked sodium hyaluronate group,the expression levels of TGF-β1 and CTGF in the Taohong Siwu jiawei decoction group were still higher(P < 0.05);Compared with the combined group,there was a slight difference in CTGF expression in Taohong Siwu jiawei decoction group and ADSCs transplantation group(P >0.05).Conclusion: 1.The IUA rat model can be successfully established by using the "double injury method".Inflammatory factors(TNF-α,IL-6 and IL-18)and fibrosis markers(TGF-β1,CTGF)may be involved in the pathogenesis of IUA in rats.2.The combination of Taohong Siwu Jiawei decoction and transplanted ADSCs can can down-regulate the expression of inflammatory factors(TNF-α,IL-6,IL-18)and fibrosis markers(TGF-β1,CTGF),which may improve fibrosis and reduce the pathological damage of IUA rats,but it cannot restore to normal level.3.The combination of Taohong Siwu Jiawei decoction and transplanted ADSCs can be comparable to crosslinked sodium hyaluronic acid,but its specific mechanism and side effects still need further study. |
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