目的:探讨左归丸(,ZGW)治疗绝经后骨质疏松症(PMOP)。
方法:通过中药系统药理学(TCMSP)筛选ZGW的主要成分和靶基因。此外,PMOP的目标基因集来自Man数据库中的GeneCards和在线孟德尔遗传。使用相邻基因的重复实例的搜索工具(STRING)11.0软件来分析相交基因之间的相互作用。采用Cytoscape3.6.1软件和Matthews相关系数(MCC)算法筛选核心基因。将50只Sprague-Dawley雌性大鼠随机分为假手术(Sham)组和四个卵巢切除(OVX)亚组。接受假手术或OVX的大鼠与载体(OVX,1毫升水/100克重量),17β-雌二醇(E2,50μg·kg-1·d-1),和ZGW冻干粉,低剂量2.3(ZGW-L)和高剂量4.6(ZGW-H)g·kg-1·d-1,连续三个月。使用双能X线和三点弯曲试验评估骨密度和骨强度。分别。此外,酶联免疫吸附测定,苏木精-伊红染色,和蛋白质印迹分析用于确定ZGW在PMOP中的潜在药理作用机制。
结果:从TCMSP中筛选出ZGW的117种活性化合物。此外,确定了108个药物和疾病的交叉基因。使用STRING软件和MCC算法,十个核心基因,包括C-X-C趋化因子活8(CXCL8),C-C趋化因子受体2型(CCR2),α-2a活性受体(ADRA2A),褪黑素受体1B型(MTNR1B),和淀粉样βA4蛋白(APP),已确定。利用Cytoscape软件构建ZGW抗骨质疏松调控网络。动物实验表明,ZGW组显著降低血清I型胶原β-C末端端肽(β-CTX)水平,升高血清骨特异性碱性磷酸酶(BALP)水平(P<0.05,P<0.01)。OVX组与Sham组比较,骨密度和骨强度均显著降低(P<0.01)。此外,用ZGW处理导致小梁厚度增加,小梁结构的改进排列,减少空骨空洞。此外,用ZGW处理显着增加CXCL8,ADRA2A,和CCR2(P<0.05,P<0.01),Runx2蛋白表达显著降低(P<0.01)。此外,ZGW和E2组BMD显著升高(P<0.05,P<0.01),提高骨强度(P<0.05,P<0.01),CXCL8、ADRA2A、和CCR2,与OVX组相比,骨组织中runt相关转录因子2水平升高(P<0.05,P<0.01)。然而,各组间MTNR1B和APP表达无显著差异。
结论:ZGW通过多种成分在PMOP中显示出协同机制,目标,和路径。
OBJECTIVE: To explore the multi-component synergistic mechanism of Zuogui Wan (, ZGW) in treating postmenopausal osteoporosis (PMOP).
METHODS: The main components and target genes of ZGW were screened via the Traditional Chinese Medicine Systems Pharmacology (TCMSP). In addition, the target gene sets of PMOP were derived from the GeneCards and Online Mendelian Inheritance in Man databases. The search tool for recurring instances of neighbouring genes (STRING) 11.0 software was used to analyze the interaction among intersecting genes. Cytoscape 3.6.1 software and the Matthews correlation coefficient (MCC) algorithm were used to screen the core genes. Fifty Sprague-Dawley female rats were randomly divided into the sham-operated (Sham) group and the four ovariectomized (OVX) subgroups. Rats subjected to Sham or OVX were administered with the vehicle (OVX, 1 mL water/100 g weight), 17β-estradiol (E2, 50 μg·kg-1·d-1), and lyophilized powder of ZGW at a low dose of 2.3 (ZGW-L) and high dose of 4.6 (ZGW-H) g·kg-1·d-1 for three months. The bone density and bone strength were assessed using dual-energy X-ray and three-point bending tests, respectively. Furthermore, enzyme-linked immun-osorbent assay, Hematoxylin-eosin staining, and western blot analysis were used to determine the potential pharmacological mechanisms of action of ZGW in PMOP.
RESULTS: A total of 117 active compounds of ZGW were screened from the TCMSP. Furthermore, 108 intersecting genes of drugs and diseases were identified. Using STRING software and the MCC algorithm, ten core genes, including C-X-C chemokine living 8 (CXCL8), C-C chemokine receptor type 2 (CCR2), alpha-2a active receptor (ADRA2A), melatonin receptor type 1B (MTNR1B), and amyloid-beta A4 protein (APP), were identified. The anti-osteoporosis regulation network of ZGW was constructed using the Cytoscape software. The animal experiments demonstrated that ZGW groups significantly reduced the serum levels of β-C-terminal telopeptide of type I collagen (β-CTX) and increased serum levels of bone-specific alkaline phosphatase (BALP) (P < 0.05, P < 0.01). The OVX group exhibited a significant decrease in bone mineral density and bone strength compared with the Sham group (P < 0.01). Moreover, treatment with ZGW resulted in increased trabecular thickness, improved arrangement of trabecular structure, and reduced empty bone lacunae. Furthermore, treatment with ZGW significantly increased the protein expression of CXCL8, ADRA2A, and CCR2 (P < 0.05, P < 0.01), and significantly decreased the protein expression of Runx2 (P < 0.01). Furthermore, the ZGW and E2 groups demonstrated significantly increased BMD (P < 0.05, P < 0.01), improved bone strength (P < 0.05, P < 0.01), reduced expression of CXCL8, ADRA2A, and CCR2, and increased runt-related transcription factor 2 levels in bone tissue (P < 0.05, P < 0.01) compared with the OVX group. However, there were no significant differences in MTNR1B and APP expression among the groups.
CONCLUSIONS: ZGW shows synergistic mechanisms in PMOP through multiple components, targets, and pathways.