To Explore The Effect And Mechanism Of Yishen Tonglong Capsule In The Treatment Of BPH Based On COX-2/PGE₂ And Related Pathways

Part 1:Estrogen and androgen synergistic induction to prepare a rat model of benign prostatic hyperplasiaObjective:To replicate the BPH rat model by using the method of estrogen and androgen synergistic induction,to study the effects of different modeling methods on prostate tissue morphology,prostate weight,prostate wet weight,prostate index,expression of b FGF and TGF-?₁in BPH rats in order to analyze its characteristics and pathological characteristics.Methods:30 SPF male SD rats were randomly divided into 6 groups:blank control group,sham operation group,non-castrated T group,non-castrated T+E group,castrated T group,and castrated group T+E group,there were 5 animals in each group.Among them,the blank control group,non-castrated T group and non-castrated T+E group did not receive any treatment.In the sham operation group,bilateral testicles were free but not removed.In castrated T group and T+E group,bilateral orchiectomy was performed.One week after the operation,the drugs were injected subcutaneously into the back and waist.The blank group and the sham operation group were given 0.9%sodium chloride injection(5mg·kg^-1),non-castrated T group and castrated T group were given testosterone propionate injection(5mg·kg^-1),non-castrated T+E group and castrated T+E group were given testosterone propionate solution(5 mg·kg^-1)combined with?-estradiol solution(0.05mg·kg^-1)for 4 weeks.After the above operation,all rats were killed by cervical dislocation method,and the prostate tissue was taken to observe the changes of prostate volume,wet weight and prostate index in each group.The pathological changes of benign prostatic hyperplasia and the expression of b FGF and TGF-?₁in anterior gland tissue were observed by HE staining and immunohistochemistry.Results:Compared with the blank group,the prostate volume,wet weight,prostate index and the expression of b FGF and TGF-?₁in the sham operation group had no significant change(P>0.05),while the prostate volume,wet weight,prostate index and the expression of b FGF in the other four groups were significantly increased(P<0.05),that is,castrated T+E group>castrated T group>non castrated T group=non castrated T+E group>sham operation group=blank group,and the castrated T+E group was the most increased;the expression of TGF-?₁was significantly decreased(P<0.05),that is,castrated T+E group<castrated T group<non castrated T group=non castrated T+E group<sham operation group=blank group,and the castrated T+E group was the most decreased.Conclusion:Both castration and non-castration methods can successfully induce BPH animal models,especially in castrated T+E group,which is most consistent with the pathological characteristics of BPH rats,and abnormal angiogenesis and inflammatory cell infiltration of prostate tissue were also accompanied in this process.It can be inferred that the imbalance of b FGF and TGF-?₁regulation is one of the pathogenesis of BPH.Part 2:The effect of Yishen Tonglong Capsule on COX-2/PGE₂and related pathways in BPH ratsObjective:To replicate the BPH rat model by using the method of estrogen and androgen synergistic induction,to observe the effect of Yishen Tonglong Capsule on the body weight,prostate volume,prostate wet weight,prostate index and prostate tissue morphology of rats with benign prostatic hyperplasia,the expression of CD3,CD20,VEGF,internal cyclooxygenase-2(COX-2)and prostaglandin E₂(PGE₂)receptors,in order to explore the mechanism of Yishen Tonglong capsules in the treatment of benign prostatic hyperplasia.Method:92 SPF male SD rats were randomly divided into 8 groups:blank group and sham operation group,10 rats in each group:model group,Longbishu group,celecoxib group,Yishen Tonglong low,middle and high dose group,12 rats in each group.The blank group did not receive any treatment.In the sham operation group,bilateral testicles were free but not removed.The other groups were treated with bilateral orchiectomy combined with subcutaneous injection of testosterone propionate and?-estradiol through the back and waist to replicate BPH rat models.After successful modeling,the blank group,sham operation group and model group were given 0.9%sodium chloride injection 10ml//kg/d by gavage,the Longbishu group was given Longbishu solution 0.24g/kg/d by gavage,the celecoxib group was given celecoxib solution 0.02g/kg/d by gavage,and the Yishen Tonglong low,middle and high dose groups were given yishentonglong solution 0.24 g,0.48g and 0.96g/kg/d by gavage respectively.After continuous administration for 8 weeks,the changes in body weight,prostate volume,wet weight,and prostate index of rats in each group were detected.The pathological changes of prostate tissue and the expression of CD3,CD20,VEGF and Ki67 in prostate tissue of rats in each group were observed by HE staining and immunohistochemistry.The microvessel density(MVD)in paraffin section of prostate tissue was calculated by Weindner method.The mRNA expression of COX-2 and PGE₂in prostate tissue was detected by real-time PCR,and the protein expression of COX-2 and PGE₂in prostate tissue was detected by Western blot.Result:Compared with the model group,the Yishen Tonglong low,middle and high dose group,the Longbishu group,and the celecoxib group can reduce the prostate volume,wet weight and prostate index of rats,and the difference is statistically significant(P<0.05),they also can significantly improve the pathological changes of prostate hyperplasia in BPH rats.Compared with the blank group,the expressions of CD3,CD20,VEGF,MVD,Ki67,COX-2,PGE₂mRNA and protein in prostate tissue of sham operation group were not significantly changed(P>0.05).while the expressions of CD3,CD20,VEGF,Ki67,COX-2,PGE₂mRNA and protein in prostate tissue of other groups were significantly increased(P<0.05).Compared with the model group,the expressions of CD3,CD20,COX-2,PGE₂mRNA and protein in prostate tissue of each drug treatment group were significantly decreased(P<0.05),especially in celecoxib group and Yishen Tonglong middle dose group(P<0.05),which had the best curative effect;the expressions of VEGF,MVD and Ki67 in prostate tissue in each drug treatment group were also significantly decreased(P<0.05),especially in Longbishu group and Yishen Tonglong middle dose group(P<0.05).Conclusion:1.The rat animal model of benign prostatic hyperplasia was successfully replicated.2.Yishen Tonglong low,middle and high dose groups,Longbishu group,and celecoxib group can reduce the prostate volume,wet weight,and prostate index of BPH rats,and improve their pathological hyperplasia.The order of curative effect is as follows:middle dose group=celecoxib group>high dose group=Longbishu group>low dose group,Yishen Tonglong middle dose group and celecoxib group had the best curative effect.3.The expression of CD3,CD20,COX-2 and PGE₂mRNA and protein in prostate tissue of BPH rats were decreased in low,middle and high dose groups of Yishen tonglong capsule,Longbishu group and celecoxib group.The order of curative effect was middle dose group=celecoxib group>high dose group=Longbishu group>low dose group.The middle dose group of Yishen tonglong capsule and celecoxib group had the best curative effect.The results indicate that both Yishen Tonglong capsule and celecoxib capsule have good anti-inflammatory effects,the mechanism of Yishen Tonglong capsule in the treatment of BPH may be the inhibition of inflammatory cells and the expression of COX-2 and PGE₂.4.The expression of MVD,VEGF and Ki76 in prostate tissue of BPH rats were decreased in low,middle and high dose groups of Yishen tonglong capsule and Longbishu group.Celecoxib had no significant effect on that.The order of curative effect was middle dose group>high dose group=Longbishu group>low dose group>celecoxib group,and the middle dose group had the best curative effect.The results showed that both Yishen Tonglong capsule and Longbishu capsule could inhibit angiogenesis and tissue proliferation.