Study On The Mechanism Of The Inhibition Of Fibroblast Activation In Rat With Uterine Leiomyoma By Sanleng-Ezhu Component Compatibility

Objectives1.To establish animal models of uterine leiomyoma（UL）successfully by combining estrogen and progesterone loading with external stimulation;2.To explore the mechanism of fibroblast-related activation of UL;3.To explore whether the effective components of Rhizome Sparganii-Curcumae Rhizoma can inhibit the fibroiblast activation of rats with UL and the mechanism of inhibiting fibroblast-related activation to prevent and treat UL.Provide new ideas for clinical treatment of uterine.MethodsThe rats were randomly divided into 9 groups:normal control group,model group,positive control group,total flavonoids group,essential oil group,S-E decoction group,S-E high dose group,S-E middle dose group,S-E low dose group,and 8 rats in each group were constructed model after 4 days of adaptive feeding.Rats in the NCG were given distilled water by gavage once a day（2 mL/day）and injected with normal saline（0.05 mL/day）through the lower limb lateral muscle 3 times a week for 5 weeks.Rats in the other 8 groups received diethylstilbestrol（1.35 mg/kg）every day,and were injected with 1.0 mg progesterone in the lateral leg on Monday,Wednesday,and Friday for 5 weeks.Then,rats were injected with 0.5mg.kg^-1.d^-1 adrenal hydrochloride from the fourth week,and given external stimulation four hours later every day（1:60 dB noise for 3 h;2:Day reversal;3:Swimming in 5–10°C water for 4 min;4:Hanging from tail for 10 min;5:Heat exposure for 10 min at 50°C）every day.Each stimulus was performed at least 2 times throughout the cycle of two weeks.From the start of modeling,the following solutions:Gong Liu Xiao solution,S-E high dose solution（66.7%solution）,S-E middle dose solution（33.3%solution）,S-E low dose solution（16.7%solution）,total flavonoids solution,essential oil solution,S-E decoction were given to the rats in positive control group,S-E high dose group,S-E middle dose group,S-E low dose group,total flavonoids group,essential oil group,S-E decoction group every afternoon.Then taken arterial blood,separated serum,weighted uterus,weighted ovary,measured the transverse and vertical diameter of uterus.Calculated the coefficients of uterus and ovary.The histopathological changes of uterus were observed by HE staining.Enzyme-linked immunosorbent assay（ELISA）was used to detect E2,P in serum.Immunohistochemistry was used to detect the expression of estrogen receptor（ER）,progesterone receptor（PR）,fibroblast activation protein（FAP）in rats uterus.Western blot was used to detect the expression of FAP,CollagenⅠ,TGF-β,Fibronectin（FN）,AKT,ERK1/2,MEK and the dephosphorylation of AKT,ERK1/2,MEK.ResultsThe rat uteruses in normal control group had pink color with no cysts,nodules,or swelling.Pathological examination showed that the epithelial cells in the uterine tissue were orderly arranged,the connective tissue in the submucosa was compact,the myometrium was orderly arranged,the myometrium was even in thickness,and the muscle fiber was clear in contour.After the modeling,the uterus of the rat showed macroscopic abnormalities,nodules and edema,and the transverse diameter and vertical diameter increased;In the model group,the uterine organ coefficient increased and the ovarian coefficient decreased;Pathological examination showed pathological changes such as uneven thickness of myometrial muscle layer,disordered muscle fiber arrangement,and fibroblast degeneration;ELISA showed that the concentration of E2（p<0.05）and P（p<0.01）in the serum of the model group rats increased;The results of immunohistochemistry showed that the expression levels of ER,PR and FAP in rat uterus were increased（p<0.05）;The results of Western blot showed that the expression of FAP（p<0.01）,CollagenI（p<0.001）,TGF-β（p<0.01）,FN（p<0.05）increased,and the phosphorylation levels of AKT（p<0.01）,ERK1/2（p<0.001）,and MEK（p<0.001）increased.After the intervention of S-E solution,which can improve the uterus malformation,nodules and edema of uterine fibroids,and also improve the pathological changes such as uneven thickness of muscle layer,disorder of muscle fiber arrangement and degeneration of fibroblasts.The transverse diameter,vertical diameter and uterine coefficient of the uterus did not change significantly in each drug-administered group（p>0.05）.The ovary coefficient in S-E middle dose group（p<0.05）,total flavonoids group（p<0.01）,essential oil group（p<0.01）improved.The E₂ concentration in serum in the S-E high dose group was decreased（p<0.05）,and the E2 concentration in the other groups was decreased,but the difference was not statistically significant（p>0.05）.P concentrations in serum in S-E high dose group（p<0.01）,S-E middle dose group（p<0.01）,and S-E low dose group（p<0.05）were decreased.P concentrations in the other groups were decreased,but the difference was not statistically significant（p>0.05）.The results of immunohistochemistry showed that the expression levels of ER protein in the uterus of the rats in the positive drug group（p<0.05）,the S-E high dose group（p<0.01）and the S-E decoction group（p<0.01）were significantly lower,and the difference was statistically significant.Although the expression of ER in the other drug-administered group decreased,the difference was not statistically significant（P>0.05）.The expression of PR protein in the uterus of the rats in the positive drug group and S-E low dose groups decreased,the difference was statistically significant（P<0.05）.The expression of PR protein in the uterus of other drug-administered group decreased,but the difference did not have statistics（P>0.05）.In positive drug group（P<0.01）,S-E high group（P<0.05）,and essential oil group（P<0.05）,the expression of FAP in uterus tissue decreased,the difference was statistically significant.The phosphorylation levels of AKT and ERK1/2 were decreased in the uterus of each administration group（P<0.05）,and the phosphorylation level of MEK was decreased in the uterus of the positive drug group,S-E high dose group,S-E middle dose group and essential oil group（P<0.05）.TGF-β1 was significantly decreased in the uterus of rats after the administration of Rhizome Sparganii total flavonoids,Curcumae Rhizoma essential oil,S-E compatibility solution,Gong Liu Xiao Capsule and S-E decoction（P<0.05）.In addition to the total flavonoids group,the expression level of Collagen I was decreased in each drug-administered group（P<0.05）,while the expression level of FN was not significantly decreased in each drug-administered group（P>0.05）.Conclusion1.The animal models of UL were constructed successfully by combining estrogen and progesterone loading;2.The S-E compatibility can reduce the content of TGF-β1 in uterus of uterine fibroids,reduce the expression of FAP,inhibit the activation of fibroblasts.3.The S-E compatibility can inhibit the activation of fibroblasts,to inhibit the expression of cell proliferation pathway factors p-AKT,p-ERK1/2,p-MEK and Collagen I,to prevent and treat UL.