A Network Pharmacology Study on the Similarities and Differences in the Mechanisms of Zuo Gui Wan/You Gui Wan for the Treatment of Premature Ovarian Failure.

BACKGROUND: Premature ovarian failure is a heterogeneous disease that severely affects the quality of life of women in their reproductive years. The ancient classical Chinese medicine compounds Zuo Gui Wan and You Gui Wan have great potential to treat premature ovarian failure, but the similarities and differences in their pharmacological mechanisms for treating POF are not yet clear. METHODS: In this study, the public database was used to screen the active ingredients and potential targets of Zuo Gui Wan and You Gui Wan. The similarities and differences in the potential targets of both pills for the treatment of POF were analysed using the POF-related genes obtained from OMIM and GeneCards. The protein-protein interaction network was established and collated to form a drug-active ingredient-target gene network using STRING. Finally, the drug-target-pathway network was constructed by enrichment analysis to find the differences in target enrichment on the same pathway. RESULTS: Pharmacological analysis of the network showed that Zuo Gui Wan contains 72 active ingredients, while You Gui Wan has 112. A total of 62 common compositions, such as quercetin and kaempferol, were identified. Amongst them were 10 unique compounds, such as hydroxyproline and cholesterol, in Zuo Gui Wan and 50 exclusive compounds, such as Karanjin and betacarotene, in You Gui Wan. In addition, 14 overlapping targets, including MAPK1, CXCL8, TNF, IL6, and EGFR, were determined amongst the first 20 targets in the treatment of POF by both pills, demonstrating that the core mechanism of POF treatment is similar between the two. Pathway enrichment analysis showed 87 identical and significant pathways between Zuo Gui Wan and You Gui Wan, including IL-17, TNF, PI3K-Akt, oestrogen, VEGF, and other pathways. Zuo Gui Wan has 12 special pathways, such as natural killer cell-mediated cytotoxicity and intestinal immune network for IgA production. You Gui Wan has nine unique pathways, such as insulin secretion and glucagon signalling pathway. CONCLUSION: Zuo Gui Wan and You Gui Wan could treat POF by inhibiting oxidative stress and inflammation, regulating hormone levels, improving ovarian function, and promoting follicular development. Zuo Gui Wan is inclined to immune regulation, while You Gui Wan prefers insulin regulation. Therefore, similarities and differences clearly exist in the specific mechanisms of Zuo Gui Wan and You Gui Wan in the treatment of POF.

Zuo Gui Wan Promotes Osteogenesis via PI3K/AKT Signaling Pathway: Network Pharmacology Analysis and Experimental Validation.

OBJECTIVE: Osteogenesis is vitally important for bone defect repair, and Zuo Gui Wan (ZGW) is a classic prescription in traditional Chinese medicine (TCM) for strengthening bones. However, the specific mechanism by which ZGW regulates osteogenesis is still unclear. The current study is based on a network pharmacology analysis to explore the potential mechanism of ZGW in promoting osteogenesis. METHODS: A network pharmacology analysis followed by experimental validation was applied to explore the potential mechanisms of ZGW in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs). RESULTS: In total, 487 no-repeat targets corresponding to the bioactive components of ZGW were screened, and 175 target genes in the intersection of ZGW and osteogenesis were obtained. And 28 core target genes were then obtained from a PPI network analysis. A GO functional enrichment analysis showed that the relevant biological processes mainly involve the cellular response to chemical stress, metal ions, and lipopolysaccharide. Additionally, KEGG pathway enrichment analysis revealed that multiple signaling pathways, including the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway, were associated with ZGW-promoted osteogensis. Further experimental validation showed that ZGW could increase alkaline phosphatase (ALP) activity as well as the mRNA and protein levels of ALP, osteocalcin (OCN), and runt related transcription factor 2 (Runx 2). What's more, Western blot analysis results showed that ZGW significantly increased the protein levels of p-PI3K and p-AKT, and the increases of these protein levels significantly receded after the addition of the PI3K inhibitor LY294002. Finally, the upregulated osteogenic-related indicators were also suppressed by the addition of LY294002. CONCLUSION: ZGW promotes the osteogenesis of BMSCs via PI3K/AKT signaling pathway.

Effect of Zuogui pill and Yougui pill on osteoporosis: a randomized controlled trial.

OBJECTIVE: To evaluate the effect and safety of Zuogui pill and Yougui pill, classic Yin and Yang tonic formula (CYYTF), in the treatment of osteoporosis and the underlying mechanism. METHODS: Participants aged 55 to 75 with osteoporosis and Kidney deficiency in Traditional Chinese Medicine (TCM) will be included and randomly allocated into two groups: treatment group and control group. Participants in the treatment group were treated with Zuogui pill or Yougui pill TCM formula granule, while the control group received placebo. Primary outcomes are the lumbar spine on bone mineral density (BMD) (L1-4) and femoral BMD. Secondary outcomes include pain intensity, health-related quality of life (HRQoL), bone turnover markers and safety. RESULTS: Totally 200 patients were enrolled from December 2014 to April 2016 from four hospitals. There were no statistically significant differences between the two groups at baseline (P > 0.05) and it was good to comparability. Statistically significant differences between the two groups were observed for the lumbar BMD (L1-4), pain VAS scores and HRQoL at six months and twelve months and femoral BMD at twelve months (P < 0.05), but no significant differences for femoral BMD and bone turnover markers at six months (P > 0.05). Moreover, significant difference was observed at different time before and after treatment in terms of lumbar spine (L1-4) BMD, femoral BMD, pain VAS scores and health-related quality of life, and there was an crossover effect between the time and groups before and after treatment. In additional, in the treatment group, 8 patients lost to follow-up and 3 patients had adverse events (AEs) and in the control group, 10 patients lost to follow-up and 2 patients had AEs. No remarkable differences were observed between the two groups with regard to AEs, lost rate and safety (P > 0.05). CONCLUSION: Zuogui pill or Yougui pill could improve BMD, ease pain, relieve Kidney deficiency syndrome, improve the quality of life osteoporosis patients, inhibit bone conversion and regulate the coupling balance of bone formation and bone resorption, but long-term efficacy should be confirmed by a longer term follow-up and larger of samples clinical randomized controlled trials.

ffects of Zuogui Pills on kidney-yin deficiency syndrome of premature ovarian insufficiency rats / 国际中医中药杂志

Objective:To study the effects of Zuogui Pills on rats with kidney-yin deficiency syndrome of premature ovarian insufficiency.Methods:Totally 40 SD female unmated rats were randomly divided into blank group, model group, Zuogui Pills group and Bujiale group, with 10 rats in each group. Except for blank group, rats in other groups were subcutaneously injected with ZP3 and gavaged with levothyroxine sodium to induce kidney-yin deficiency syndrome model of premature ovarian insufficiency. At the same time of modeling, Zuogui Pills group and Bujiale group received corresponding drugs for gavage, and the other groups received corresponding solvent for gavage, once a day, for consecutive 21 days. On day 0, 7, 14 and 21, ear temperature and body weight of rats were measured, and the ovarian index, uterus index and thyroid index were calculated. Serum levels of adenosine cyclic phosphate (cAMP), cyclic guanosine phosphate (cGMP), Cortisol, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E 2) were detected by ELISA. Pathological changes of ovary was observed with HE staining. Results:On day 14 and 21, compared with model group, the body weight of rats in Zuogui Pills group increased ( P<0.05), and the ear temperature decreased ( P<0.05); compared with model group, the ovarian index, uterine index and thyroid index of rats in Zuogui Pills group decreased ( P<0.05), the levels of serum cAMP/cGMP, cortisol, FSH and LH decreased ( P<0.05), and the level of E 2 increased ( P<0.05). Conclusion:Zuogui Pills have certain improvement effect on rats with kidney-yin deficiency syndrome induced by levothyroxine sodium tablets combined with ZP3.

Effects of Zuogui Pill on osteoclast activity and expression of miR-133b-3p/RhoA in postmenopausal osteoporosis rats / 国际中医中药杂志

Objective:To study the effects of Zuogui Pills on the expressions of miR-133b-3p and RhoA in osteoclasts of postmenopausal osteoporosis rats; To discuss its potential mechanism.Methods:SD female rats were randomly divided into normal group, model group, sham-operation group, and Zuogui Pills group using a random number table method, with 6 rats in each group. The model group and Zuogui Pills group were treated with oophorectomy to construct a rat model of osteoporosis. Zuogui Pills group was orally administered with Zuogui Pills decoction at a concentration of 10 g/kg for 12 consecutive weeks. Colorimetric method was used to measure the serum calcium and phosphorus levels of rats, and ELISA method was used to detect ALP levels. Bone density meter was used to measure the bone density of the femurs of rats in each group. The osteoclast of each group were cultured, and the expressions of RANKL and RUNX2 protein were detected by Western blot. MiRNA sequencing and differential expression analysis were performed on bone tissues of rats. Osteoclasts were treated with miR-133b-3p mimic and its negative control. The cell proliferation activity of osteoclasts was detected by cell counting kit-8 (CCK-8). The osteoclast differentiation activity was detected by the tartrate-resistant acid phosphatase staining. The dual-luciferase reporter assay was used to detect the relationship between miR-133b-3p and RhoA. The "rescue" experiment of miR-133b-3p mimic and RhoA co-expression were used to study the molecular regulatory mechanism of Zuogui Pills on osteoclast activity.Results:Compared with the model group, the bone mineral density of Zuogui Pills group significantly increased ( P<0.05, P<0.01), the levels of calcium and phosphorus in serum increased, the level of alkaline phosphatase ALP decreased ( P<0.05), the expression of RANKL protein decreased, and the expression of RUNX2 protein increased. Sequencing results showed that rno-miR-133b-3p was down-regulated in osteoclasts of postmenopausa osteoporosis rats treated with Zuogui Pills with the maximum difference ( P<0.01). Q-PCR results showed that the expression of miR-133b-3p in osteoclasts of Zuogui Pills group was significantly lower than that of the model group. The upregulation of miR-133b-3p could significantly promote the cell proliferation and differentiation of osteoclasts. RhoA overexpression could reverse the excessive proliferation and differentiation of osteoclasts caused by miR-133b-3p overexpression. Conclusions:RhoA is the target gene regulated by miR-133b-3p. Zuogui Pills can inhibit the activity of osteoclasts by regulating miR-133b-3p/RhoA axis, relieving the symptoms of osteoporosis.

Regulatory effect of traditional Chinese medicinal formula Zuo-Gui-Wan on the Th17/Treg paradigm in mice with bone loss induced by estrogen deficiency.

ETHNOPHARMACOLOGICAL RELEVANCE: Bone loss is a common pathological condition induced by estrogen deficiency. The Th17/Treg paradigm, which can be skewed by estrogen, plays an important role in regulating bone metabolism AIM OF THE STUDY: The purpose of this study was to determine the role of the Th17/Treg shift in estrogen deficiency-induced bone loss in mouse models and to elucidate the immunopharmacologic mechanism of Zuo-Gui-Wan (ZGW) in preventing bone loss in this process by regulating Th17/Treg paradigm. MATERIALS AND METHODS: Splenocytes of ovariectomized (Ovx) mice and naturally aged mice were isolated and Flow cytometry was used to detect the Th17/Treg subsets. In addition, serum estrodiol (E2) and serum C-terminal telopeptides of type Ι collagen (CTx) were detected by ELISA assay. Bone mineral density (BMD) of the left tibiae was measured by dual-energy X-ray absorptiometry. Moreover, Ovx mice were administrated with different doses of ZGW for 12 weeks, and BMD and Th17/Treg subsets were determined. Bone histomorphometry was observed by Hematoxylin and eosin (H&E) staining and serum protein levels of IL-6 were analyzed by ELISA assay. In addition, the mRNA and protein expression of RORγt and Foxp3 were detected by RT-PCR and Western blot respectively. RESULT: The Th17/Treg paradigm shifted to Th17 in estrogen-deficient mice both in the Ovx mice and the naturally aged mice. BMD and E2 levels negatively correlated with the Th17/Treg ratio. After ZGW administration, the BMD was enhanced markedly in the Ovx mice as well as in the naturally aged mice. Both the mRNA and protein expressions of IL-6 and RORγt were decreased, whereas those of Foxp3 were increased significantly after ZGW administration. CONCLUSION: Th17/Treg shift involved in the bone loss induced by estrogen deficiency. ZGW prevented bone loss efficiently and skewed Th17/Treg paradigm towards Treg without enhancing E2.