中药药理与临床

目的:观察茱萸丸对雄激素诱导的金黄地鼠皮脂溢出的影响及其相关机制。方法:采用双氢睾酮诱导金黄地鼠耳廓组织建立皮脂溢出动物模型，总造模周期为18 d。造模成功的50只金黄地鼠随机分为模型对照组、茱萸丸106、212、425 mg/kg及异维A酸3.7 mg/kg组，每组10只，另设正常对照组10只。造模后各组灌胃给予相应药物或无菌蒸馏水，连续4 w。评价耳廓形态学与HE病理改变；生化法检测耳廓组织游离脂肪酸(NEFA)、甘油三酯(TG)含量；ELISA法检测血清中睾酮(T)、雌二醇(E2)、促黄体生成素(LH)含量；16S rDNA技术检测金黄地鼠粪便的肠道菌群Alpha、Beta多样性及物种组成差异；UHPLC-MS/MS检测金黄地鼠血浆中氧化三甲胺(TMAO)、三甲胺(TMA)含量；Pearson检验分析肠道菌群与TMAO、TMA的相关性；免疫组化法检测金黄地鼠耳廓组织核苷酸结合寡聚化结构域样受体家族吡啉结构域蛋白3 (NLRP3)、含CARD结构域的凋亡相关颗粒样蛋白(ASC)、含半胱氨酸的天冬氨酸蛋白酶1 (Caspase-1)蛋白表达水平；Western blot及Real-time PCR法检测耳廓组织髓样分化因子88(MyD88)、白细胞介素1受体相关激酶1(IRAK1)、肿瘤坏死因子受体相关因子6(TRAF6)、核转录因子κB p65(NF-κB p65)蛋白及mRNA表达。结果:与正常对照组相比，模型对照组耳廓皮肤增厚、角化过度，血清T、LH含量升高，E2含量降低，肠道菌群Alpha多样性下降，厚壁菌门、杜氏牙形石属丰度升高，拟杆菌门、放线菌门、圣埃里立支杆菌属、粪杆菌属、布劳特氏菌属丰度降低，血浆TMAO、TMA、NLRP3炎性小体相关蛋白以及MyD88/IRAK1/TRAF6/NF-κB通路基因和蛋白表达上调(P<0.01);与模型对照组比较，茱萸丸各剂量组耳廓病变改善，血清T、LH含量降低、E2含量升高，肠道菌群Alpha多样性升高，疣微菌门、拟杆菌门、圣埃里立支杆菌属、布劳特氏菌属、嗜黏蛋白阿克曼菌属丰度升高，厚壁菌门、杜氏牙形石属丰度降低，血浆TMAO、TMA含量降低，NLRP3炎性小体及MyD88/IRAK1/TRAF6/NF-κB通路蛋白与mRNA表达下调(P<0.05或P<0.01)。结论:茱萸丸能够调控性激素水平，抑制皮脂溢出的病理改变，其机制可能与纠正菌群紊乱，减少TMAO、TMA生成，调控MyD88/IRAK1/TRAF6/NF-κB信号通路及其下游NLRP3炎性小体的表达有关。

Objective:This paper aims to observe the effect of Zhuyu(茱萸) Pill on sebum secretion induced by androgen in golden hamsters and its related mechanism. Methods:An animal model of sebum secretion was established by using dihydrotestosterone to induce the auricular tissue of golden hamsters. The total modeling period was 18 days. 50 successfully modeling golden hamsters were randomly divided into a model control group, Zhuyu Pill groups at doses of 106,212,and 425 mg/kg, and an isotretinoin group at a dose of 3.7 mg/kg, with 10 hamsters in each group. Another 10 hamsters were selected as the normal control group. After modeling, the normal control group and the model control group were given the same volume of sterile distilled water by gavage for four weeks. The pathological changes of auricular morphology and hematoxylin-eosin(HE) staining were evaluated. The contents of non-esterified fatty acid(NEFA) and triacylglycerol(TG) in the auricular tissue were detected by biochemical methods. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of testosterone(T),estradiol(E2),and luteinizing hormone(LH) in the serum. 16S rDNA technology was used to detect the differences in Alpha and Beta diversity and species composition of the gut microbiota in the feces of golden hamsters. UHPLC-MS/MS was used to detect the contents of trimethylamine oxide(TMAO) and trimethylamine(TMA) in the plasma of golden hamsters. Pearson was used to measure and analyze the correlation between gut microbiota and TMAO and TMA. Immunohistochemistry was used to detect the protein expression levels of NOD-like receptor family pyrin domain containing 3(NLRP3),apoptosis-associated speck-like protein containing CARD(ASC),and cysteinyl aspartate specific protease 1(Caspase-1) in the auricular tissue of golden hamsters. Western blot and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) were used to detect the protein and mRNA expression levels of myeloid differentiation primary response protein 88(MyD88),interleukin-1 receptor-associated kinase 1(IRAK1),tumor necrosis factor receptor-associated factor 6(TRAF6),and nuclear factor kappa-B p65(NF-κB p65). Results:Compared with the normal control group, the model control group showed thickened auricular skin, excessive keratinization, elevated serum T and LH levels, decreased E2 levels, decreased Alpha diversity of gut microbiota, increased Firmicutes and Dubosiella,decreased Bacteroidota, Actinobacteria, Ligilactobacillus,Faecaculum,and Blautia,as well as increased expression levels of TMAO,TMA,NLRP3 inflammasome, and MyD88/IRAK1/TRAF6/NF-κB pathways in the plasma(P<0.01). Compared with those in the model control group, the auricular lesions in groups with different doses of Zhuyu Pill were improved, the levels of T and LH were decreased in the serum, the E2 level was increased. Alpha diversity of gut microbiota was increased, the Verrucomicrobiota, Bacteroidota, Ligilactobacillus,Blautia, and Akkermansia were increased, while the Firmicutes and Dubosiella were decreased. The levels of TMAO and TMA in the plasma were decreased, and the protein and mRNA expressions of NLRP3 inflammasome and MyD88/IRAK1/TRAF6/NF-κB pathways were decreased(P<0.05 or P<0.01). Conclusion:Zhuyu Pill can regulate the levels of sex hormones and inhibit the pathological changes of sebum excretion. Its mechanism may be related to correcting the imbalance of the microbiota, reducing the production of TMAO and TMA,regulating the MyD88/IRAK1/TRAF6/NF-κB signaling pathways and the expression of its downstream NLRP3 inflammasome.