目的 探究五子衍宗丸（WZYZP）治療少弱精子癥（OAS）的藥效與作用機(jī)制。方法 應(yīng)用環(huán)磷酰胺（CTX）誘導(dǎo)的SD大鼠作為OAS的體內(nèi)模型，通過ig 0.5、1.5 g·kg^-1 WZYZP探究其治療OAS大鼠的體內(nèi)藥效與作用機(jī)制。通過全自動(dòng)精子分析系統(tǒng)檢測(cè)大鼠精子參數(shù)；應(yīng)用酶聯(lián)免疫吸附法測(cè)定大鼠血清中睪酮的量，通過HE染色法觀察給藥后OAS大鼠睪丸組織形態(tài)學(xué)改變。采用網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)WZYZP治療OAS的具體靶點(diǎn)。應(yīng)用酶聯(lián)免疫吸附法測(cè)定大鼠睪丸組織炎癥因子白細(xì)胞介素1β（IL-1β）、白細(xì)胞介素6（IL-6）和腫瘤壞死因子α（TNF-α）水平；通過TUNEL染色測(cè)定大鼠睪丸組織中細(xì)胞凋亡情況；免疫組織化學(xué)染色檢測(cè)睪丸組織白細(xì)胞介素17（IL-17）表達(dá)情況；Western blotting檢測(cè)Caspase-3、熱休克蛋白90AA1（HSP90AA1）、磷酸化核因子κB（p-NF-κB）、核因子κB（NF-κB）、磷酸化細(xì)胞外調(diào)節(jié)蛋白激酶1/2（p-ERK1/2）、細(xì)胞外調(diào)節(jié)蛋白激酶1/2（ERK1/2）、p-p38和p38的表達(dá)水平。使用HPLC檢測(cè)WZYZP中的主要活性成分并與網(wǎng)絡(luò)藥理學(xué)所得靶點(diǎn)進(jìn)行分子對(duì)接模擬以探索其具體機(jī)制。結(jié)果 體內(nèi)實(shí)驗(yàn)結(jié)果表明，WZYZP可顯著改善 CTX誘導(dǎo)的 OAS大鼠精子參數(shù)、血清睪酮水平（P＜0.001）和睪丸組織形態(tài)學(xué)。網(wǎng)絡(luò)藥理學(xué)結(jié)果表明WZYZP與OAS的關(guān)鍵通路為IL-17，關(guān)鍵靶點(diǎn)為 Caspase3、p38、NF-κB、HSP90和 ERK1/2。TUNEL染色結(jié)果表明，1.5 g·kg^-1 WZYZP可顯著降低 OAS大鼠睪丸組織細(xì)胞凋亡 （P＜0.05）。 Western Blotting結(jié)果顯示 ，與模型組相比 ， 1.5 g·kg^-1WZYZP組 Caspase-3 （P < 0.001）和HSP90AA1（P < 0.01）的表達(dá)水平明顯降低，NF-κB（P < 0.05）、ERK1/2（P < 0.05）和 p38（P < 0.05）磷酸化水平明顯降低。分子對(duì)接模擬結(jié)果發(fā)現(xiàn)，8種主要活性成分（槲皮素、五味子醇甲、金絲桃苷、山柰酚、紫云英苷、五味子甲素、異槲皮素和綠原酸）和IL-17通路5種相關(guān)靶點(diǎn)（HSP90AA1、ERK、p38、NF-κB和Caspase3）的結(jié)合能均小于-20.92 kJ·mol^-1，具有較好的結(jié)合親和力。結(jié)論 WZYZP可通過下調(diào)IL-17通路減少細(xì)胞凋亡發(fā)揮治療OAS作用。

Objective To investigate the efficacy and mechanism of action of Wuziyanzong Pill (WZYZP) in treatment of oligoasthenozoospermia (OAS).Methods Cyclophosphamide (CTX)-induced SD rats were used as an in vivo model of OAS, and the in vivo efficacy and mechanism of action of WZYZP were investigated by ig 0.5 and 1.5 g·kg^-1 WZYZP. The sperm parameters of rats were detected by automatic sperm analysis system; the amount of testosterone in serum of rats was measured by enzymelinked immunosorbent assay, and the histomorphometric changes of testes of OAS rats were observed by HE staining after drug administration. Network pharmacology was used to predict the specific targets of WZYZP in the treatment of OAS. The levels of inflammatory factors interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) in rat testicular tissue were determined by enzyme-linked immunosorbent assay (ELISA); apoptosis in rat testicular tissue was determined by TUNEL staining; the expression of interleukin 17 (IL-17) in testicular tissue was detected by immunohistochemical staining; and the expression of cysteine-containing cells in testicular tissue was detected by western blotting. Blotting was performed to detect the expression of Caspase-3, heat shock protein 90AA1 (HSP90AA1), phosphorylated nuclear factor κB (p-NF- κB), nuclear factor κB (NF- κB), phosphorylated extracellular regulated protein kinase 1/2 (p-ERK1/2), ERK1/2, p-p38 and p38 expression levels. The main active components in WZYZP were detected using HPLC and molecular docking simulations with targets obtained from network pharmacology were performed to explore the specific mechanisms.Results The results of in vivo experiments showed that WZYZP significantly improved sperm parameters, serum testosterone levels (P < 0.001) and testicular histomorphometry in CTX-induced OAS rats. The results of network pharmacology showed that the key pathway of WZYZP with OAS was IL-17, and the key targets were Caspase-3, p38, NF-κB, HSP90 and ERK1/2. TUNEL staining results showed that 1.5 g·kg^-1 WZYZP significantly reduced apoptosis in testicular tissues of OAS rats (P < 0.05); Immunohistochemistry results showed that 1.5 g·kg^-1WZYZP significantly reduced testicular IL-17 expression in OAS rats (P < 0.001); ELISA results showed that IL-1β (P < 0.05) in the testicular tissues of rats in the 1.5 g·kg^-1 WZYZP group compared with the model group, IL-6 (P < 0.01) and TNF-α (P < 0.01) were significantly lower in the testicular tissues of rats in the 1.5 g·kg^-1 WZYZP group compared with the model group; the results of Western Blotting showed that the expression levels of Caspase-3 (P < 0.001) and HSP90AA1 (P < 0.01) were significantly lower in the 1.5 g·kg^-1 WZYZP group compared with the model group, and the expression level of NF-κB (P < 0.05), ERK1/2 (P < 0.05) and p38 (P < 0.05) phosphorylation levels were significantly reduced. Molecular docking simulations revealed that the binding energies of the eight main active ingredients (quercetin, schisandrin A, chrysin, kaempferol, zingiber officinale, schisandrin, isoquercitrin, and chlorogenic acid) and the five relevant targets of the IL-17 pathway (HSP90AA1, ERK, p38, NF-κB, and Caspase3) were all less than -20.92 kJ·mol^-1, with good binding.Conclusion WZYZP can reduce apoptosis by down-regulating IL-17 pathway to treat OAS.

R965

南通市科技計(jì)劃項(xiàng)目（JC2021011）