Revealing the mechanisms of Weishi Huogu I capsules used for treating osteonecrosis of the femoral head based on systems pharmacology with one mechanism validated with in vitro experiments.

ETHNOPHARMACOLOGICAL RELEVANCE: Weishi Huogu I (WH I) capsules, developed through traditional Chinese medicine, have been used to treat clinical osteonecrosis of the femoral head (ONFH) for decades. However, the mechanisms have not been systematically studied. AIM OF THE STUDY: In this study, the mechanisms of WH I capsules used in treating ONFH were examined through a systems pharmacology strategy, and one mechanism was validated with in vitro experiments. MATERIALS AND METHODS: WH I capsules compounds were identified by screening databases; then, a database of the potential active compounds was constructed after absorption, distribution, metabolism and excretion (ADME) evaluation. The compounds were identified through a systematic approach in which the probability of an interaction of every candidate compound with each corresponding target in the DrugBank database was calculated. Gene Ontology (GO) and pathway enrichment analyses of the targets was performed with the Metascape and KEGG DISEASE databases. Then, a compound-target network (C-T) and target-pathway network (T-P) of WH I capsule components were constructed, and network characteristics and related information were used for systematically identifying WH I capsule multicomponent-target interactions. Furthermore, the effects of WH I capsule compounds identified through the systematic pharmacology analysis of the osteogenic transformation of human umbilical mesenchymal stem cells (HUMSCs) were validated in vitro. RESULTS: In total, 152 potentially important compounds and 176 associated targets were identified. Twenty-two crucial GO biological process (BP) or pathways were related to ONFH, mainly in regulatory modules regulating blood circulation, modulating growth, and affecting pathological processes closely related to ONFH. Furthermore, the GO enrichment analysis showed that corydine, isorhamnetin, and bicuculline were enriched in "RUNX2 regulates osteoblast differentiation", significantly increased alkaline phosphatase activity and calcium deposition and upregulated runt-related transcription factor 2 mRNA and protein expression and osteocalcin mRNA expression in HUMSCs, suggesting that these compounds promoted the mesenchymal stem cell (MSC) osteogenic transformation. CONCLUSIONS: The study showed that the pharmacological mechanisms of WH I capsule attenuation of ONFH mainly involve three therapeutic modules: blood circulation, modulating growth, and regulating pathological processes. The crosstalk between GOBPs/pathways may constitute the basis of the synergistic effects of the compounds in WH I capsules in attenuating ONFH. One of the pharmacological mechanisms in the WH I capsule effect on ONFH involves enhancement of the osteogenic transformation of MSCs, as validated in experiments performed in vitro; however, more mechanisms should be validated in further studies.

4-Hydroxyisoleucine relieves inflammation through iRhom2-dependent pathway in co-cultured macrophages and adipocytes with LPS stimulation.

BACKGROUND: 4-Hydroxyisoleucine (4-HIL) is an active ingredient extracted from Trigonella foenum-graecum L., a Chinese traditional herbal medicine, which exerts the efficacy of anti-obesity and anti-diabetes. We previously reported that 4-HIL potentiates anti-inflammatory and anti-insulin resistance effects through down-regulation of TNF-α and TNF-α converting enzyme (TACE) in 3 T3-L1 adipocytes and HepG2 cells. In the present study, we further investigate the effects and mechanisms of 4-HIL on obesity-induced inflammation in RAW264.7 macrophages and 3 T3-L1 adipocytes co-culture system. METHODS: RAW264.7 macrophages and 3 T3-L1 adipocytes were co-cultured to mimic the microenvironment of adipose tissue. siRNA-iRhom2 transfection was performed to knockdown iRhom2 expression in RAW264.2 macrophages. The mRNA and protein expression of iRhom2 and TACE were measured by real-time quantitative PCR (RT-qPCR) and western blotting. The production of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), IL-6 and IL-10 were evaluated by ELISA. The ratio of M2/M1 was detected by flow cytometry. RESULTS: 4-HIL significantly repressed the mRNA and protein levels of iRhom2 and TACE in RAW264.7 macrophages after LPS stimulated. Meanwhile, the levels of pro-inflammatory cytokines, including TNF-α, MCP-1, and IL-6, were substantially suppressed by 4-HIL in the co-culture system. Moreover, the level of anti-inflammatory cytokine IL-10 was increased significantly by 4-HIL in the co-culture system after LPS stimulation. Additionally, the ratio of M2/M1 was also increased by 4-HIL in the co-culture system after LPS stimulation. Finally, these effects of 4-HIL were largely enhanced by siRNA-iRhom2 transfection. CONCLUSION: Taken together, our results indicated that obesity-induced inflammation was potently relieved by 4-HIL, most likely through the iRhom2-dependent pathway.

Fenugreek attenuates obesity-induced inflammation and improves insulin resistance through downregulation of iRhom2/TACE.

AIMS: We previously reported that fenugreek-derived 4-hydroxyisoleucine ameliorates insulin resistance via regulation of TNF-α converting enzyme (TACE) expression. In the present study, we further investigate the effects and mechanisms of fenugreek on obesity-induced inflammation and insulin signaling in the high-fat diet (HFD)-challenged obese mice. MAIN METHODS: After 12 weeks of HFD intervention, mice were treated with the low or high dosages of fenugreek. Serum levels of glucose, insulin, lipid profile, inflammation cytokines, and adipokines were detected. Macrophage infiltration and adipose tissue morphology were observed. Western blot was conducted to investigate the expressions of inactive rhomboid 2 (iRhom2) and TACE as well as other signaling pathways in subcutaneous adipose tissue. KEY FINDINGS: We showed that fenugreek significantly suppressed body weight gain and fat accumulation in HFD-challenged obese mice. Meanwhile, fasting glucose, insulin, and HOMA-IR in fenugreek-treated mice were remarkably decreased, which were properly explained by fenugreek-induced activation of the insulin receptor signaling pathway. Moreover, the anti-inflammatory properties of fenugreek were shown by the decrease of systemic and local expressions of pro-inflammatory cytokines as well as reduced macrophage infiltration into adipose tissue. Additionally, fenugreek markedly deactivated NF-κB and JNK pathways. Finally, we demonstrated that fenugreek strikingly repressed the transcriptions and expressions of iRhom2 and TACE. SIGNIFICANCE: Fenugreek shows an encouraging and promising property in ameliorating insulin resistance and suppressing inflammation in obesity, which might be realized by fenugreek-mediated inhibition of iRhom2/TACE axis-facilitated TNF-α release from adipocytes.

The protective effect of resveratrol on islet insulin secretion and morphology in mice on a high-fat diet.

The aim of this study was to investigate the effect of resveratrol on beta cells in male C57BL/6J mice fed a high-fat diet and the possible mechanisms. Male C57BL/6J mice were randomly divided into three groups: normal control (NC) group, high-fat diet (HF) group and high-fat diet and resveratrol treatment (HFR) group (15 in each group). HFR group was fed with high fat diet for 8 weeks and then orally administered resveratrol at 400mg/kg daily. Twenty-four weeks later, the function of insulin secretion in vivo and in vitro was improved robustly in HFR group compared with HF group. The levels of glucose and lipid metabolism, beta cell mass, lipid content, and oxidative stress were lower in HFR group than in HF group. Simultaneously, resveratrol administration promoted the expression of SIRT1 in islets, while the expression of uncoupling protein 2 (UCP2) was restrained. Resveratrol, as well, also had a beneficial effect on the ratios of expressions of Bcl-2/Bax and levels of malondialdehyde/glutathione peroxidase. Resveratrol can protect islets from abnormal insulin secretion and morphological changes induced by a high-fat diet. The effect might be partly related to activated SIRT1 signal pathway, improved oxidative stress induced damage and incidence of apoptosis.

Insulin improves ß-cell function in glucose-intolerant rat models induced by feeding a high-fat diet.

Insulin therapy has been shown to contribute to extended glycemia remission in newly diagnosed patients with type 2 diabetes mellitus. This study investigated the effects of insulin treatment on pancreatic lipid content, and ß-cell apoptosis and proliferation in glucose-intolerant rats to explore the protective role of insulin on ß-cell function. A rat glucose-intolerant model was induced by streptozotocin and a high-fat diet. Plasma and pancreatic triglycerides, free fatty acids, and insulin were measured; and pancreatic ß-cell cell apoptosis and proliferation were detected by a propidium iodide cell death assay and immunofluorescence for proliferating cell nuclear antigen. Relative ß-cell area was determined by immunohistochemistry for insulin, whereas insulin production in pancreas was assessed by reverse transcriptase polymerase chain reaction. Islet ß-cell secreting function was assessed by the index ΔI30/ΔG30. Glucose-intolerant rats had higher pancreatic lipid content, more islet ß-cell apoptosis, lower ß-cell proliferation, and reduced ß-cell area in pancreas when compared with controls. Insulin therapy reduced blood glucose, inhibited pancreatic lipid accumulation and islet ß-cell apoptosis, and increased ß-cell proliferation and ß-cell area in glucose-intolerant rats. Furthermore, impaired insulin secretion and insulin production in glucose-intolerant rats were improved by insulin therapy. Insulin can preserve ß-cell function by protecting islets from glucotoxicity and lipotoxicity. It can also ameliorate ß-cell area by enhancing ß-cell proliferation and reducing ß-cell apoptosis.