Effects of resveratrol in an animal model of osteoporosis: a meta-analysis of preclinical evidence.

Background: Resveratrol is a natural polyphenol compound that is widely present in herbal medicines such as Reynoutria japonica Houtt., Veratrum nigrum L., and Catsiatora Linn and is used in traditional Chinese medicine to treat metabolic bone deseases. Animal experiments have shown that resveratrol may have a strong treatment effect against osteoporosis (OP). The purpose of this study was to explore the efficacy of resveratrol in treating OP animal models based on preclinical research data. Methods: This study was completed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We searched the PubMed, Embase, Cochrane Library, and China National Knowledge Infrastructure (CNKI) databases from inception to May 8, 2023, to identify animal experiments on the treatment of OP with resveratrol. The effect sizes of bone mineral density (BMD), parameters of micro-CT, serum calcium, phosphorus, alkaline phosphatase (ALP) and osteocalcin were expressed as the mean differences (MDs) and 95% confidence intervals (CIs). RevMan 5.4 software was used for data analysis. Results: This meta-analysis included a total of 15 animal experiments, including 438 OP rats. The meta-analysis results showed that compared with the control group, resveratrol (<10, 10-25, 40-50, ≥ 60 mg/kg/day) significantly increased femoral and lumbar bone mineral density (BMD) in OP rats (p < 0.05). Resveratrol (<10 mg/kg/day) significantly increased the BMD of the total body (MD = 0.01, 95% CI: 0.01 to 0.01, p < 0.001). In terms of improving the parameters related to micro-CT, resveratrol (40-50 mg/kg/day) can increase trabecular thickness and trabecular number and reduce trabecular spacing (p < 0.05). Compared with the control group, resveratrol can reduce the concentration of calcium and phosphorus in serum but has no significant effect on serum ALP and osteocalcin (p > 0.05). The results of subgroup analysis showed that resveratrol increased the whole-body BMD of SD rats (p = 0.002) but did not improve the whole-body BMD of 3-month-old rats (p = 0.17). Conclusion: Resveratrol can increase BMD in OP rat models, and its mechanism of action may be related to improving bone microstructure and regulating calcium and phosphorus metabolism. The clinical efficacy of resveratrol in the treatment of OP deserves further research.

The Protective Effect of the Asystasia chelonoides Extracts on Hypertensive Nephropathy Rats.

BACKGROUND: Hypertensive nephropathy (HN) is one kind of kidney disorders caused by long-term uncontrolled hypertension, usually resulting in severe kidney damage, including inflammation and oxidative stress, no matter in cells or tissues, from patients with nephropathy. In recent years, nephropathy accompanied by hypertension is becoming one of the main causes for kidney replacement therapy, but few effective treatments have been reported for HN treatment. Asystasia chelonoides (AC) is a kind of plant with the effects of anti-inflammation, lowering blood pressure, and anti-oxidative stress. Still, the therapeutic effect of AC in HN rats is not clear. METHODS: To establish HN model by feeding high sugar and high fat diet spontaneously hypertensive rats. Blood measurement, HE staining, PAS staining and biochemical analysis and were used to assess the therapeutic effects of AC extracts and western blotting analyzed the underlying mechanisms of AC extracts treatment in the HN rat model. RESULTS: AC extracts could significantly lower systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean blood pressure (MBP) in HN rats; and reduce the expression of total protein (TP), blood urea nitrogen (BUN), microalbuminuria (MALB), creatinine (Cr), total cholesterol (TC), triglyceride (TG), and low-density lipoprotein-cholesterol (LDL-C) concentrations, and also could down-regulate expression of IL-6, MDA and AGEs, up-regulate the expression of SOD in HN rats; HE staining and PAS staining demonstrated that AC extracts could alleviate the histopathological changes in HN rats; western blotting demonstrated that AC extracts could up-regulate the expression of PPARγ and down-regulate the expression of TGFß1 and NF-кB in HN rats. CONCLUSION: The finding of the article demonstrated that AC extracts had the better therapeutic effect for HN, and provided the pharmacological evidences for AC extracts treatment for HN.

Moshen granule ameliorates membranous nephropathy by blocking intrarenal renin-angiotensin system signalling via the Wnt1/ß-catenin pathway.

BACKGROUND: Membranous nephropathy (MN) is one of the cardinal causes of nephrotic syndrome in adults, but an adequate treatment regimen is lacking. PURPOSE: We assessed the effect of Moshen granule (MSG) on patients with MN and cationic bovine serum albumin (CBSA)-induced rats. We further identified the bioactive components of MSG and revealed the underlying molecular mechanism of its renoprotective effects. METHODS: We determined the effect of MSG on patients with MN and CBSA-induced rats and its components on podocyte injury in zymosan-activated serum (ZAS)-elicited podocytes and revealed their regulatory mechanism on the Wnt/ß-catenin/renin-angiotensin system (RAS) signalling axis. RESULTS: MSG treatment improved renal function and reduced proteinuria in MN patients and significantly reduced proteinuria and preserved the protein expression of podocin, nephrin, podocalyxin and synaptopodin in CBSA-induced MN rats. Mechanistically, MSG treatment significantly inhibited the protein expression of angiotensinogen, angiotensin converting enzyme and angiotensin II type 1 receptor, which was accompanied by inhibition of the protein expression of Wnt1 and ß-catenin and its downstream gene products, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in CBSA-induced MN rats. We further identified 81 compounds, including astragaloside IV (AGS), calycosin, barleriside A and geniposidic acid, that preserve the podocyte-specific protein expression in ZAS-induced podocytes. Among these four compounds, AGS exhibited the strongest inhibitory effects on podocyte protein expression. AGS treatment significantly inhibited the protein expression of RAS components and Wnt1 and ß-catenin and its downstream gene products in ZAS-induced podocytes. In contrast, the inhibitory effect of AGS on podocyte-specific proteins, ß-catenin downstream gene products and RAS components was partially abolished in ZAS-induced podocytes treated with ICG-001 and ß-catenin siRNA. CONCLUSION: This study first demonstrates that AGS mitigates podocyte injury by inhibiting the activation of RAS signalling via the Wnt1/ß-catenin pathway by both pharmacological and genetic methods. Therefore, AGS might be considered a new ß-catenin inhibitor that inhibits the Wnt1/ß-catenin pathway to retard MN in patients.

Quercetin protects against iron overload-induced osteoporosis through activating the Nrf2/HO-1 pathway.

AIMS: Eucommia is the tree bark of Eucommia japonica, family Eucommiaceae. In traditional Chinese medicine, Eucommia is often used to treat osteoporosis. Quercetin (QUE), a major flavonoid extract of Eucommia japonica, has been reported to have anti-osteoporosis effects. However, there are no studies reporting the mechanism of QUE in the treatment of iron overload-induced osteoporosis. This study set out to investigate the therapeutic effects of QUE against iron overload-induced bone loss and its potential molecular mechanisms. MATERIALS AND METHODS: In vitro, MC3T3-E1 cells were used to study the effects of QUE on osteogenic differentiation, anti-apoptosis and anti-oxidative stress damage in an iron overload environment (FAC 200 µM). In vivo, we constructed an iron overload mouse model by injecting iron dextrose intraperitoneally and assessed the osteoprotective effects of QUE by Micro-CT and histological analysis. KEY FINDINGS: In vitro, we found that QUE increased the ALP activity of MC3T3-E1 cells in iron overload environment, promoted the formation of bone mineralized nodules and upregulated the expression of Runx2 and Osterix. In addition, QUE was able to reduce FAC-induced apoptosis and ROS production, down-regulated the expression of Caspase3 and Bax, and up-regulated the expression of Bcl-2. In further studies, we found that QUE activated the Nrf2/HO-1 signaling pathway and attenuated FAC-induced oxidative stress damage. The results of the in vivo study showed that QUE was able to reduce iron deposition induced by iron dextrose and attenuate bone loss. SIGNIFICANCE: Our results suggested that QUE protects against iron overload-induced osteoporosis by activating the Nrf2/HO-1 signaling pathway.

The Protective Effect of Bajijiasu on the Treatment of Hypertensive Nephropathy in Rats.

BACKGROUNDS: Hypertensive nephropathy (HN) is a kind of renal disease caused by essential hypertension that eventually worsens into end-stage renal disease (ESRD). HN could damage the renal tubules, induce kidney damage and renal failure, and increase the risk of stroke, heart disease or death, but there are few ideal drugs for HN treatment. METHODS: In this study, we explored the therapeutic effect of bajijiasu (a compound from Morinda officinalis how and a common traditional Chinese medicine for tonifying the kidney) on the HN rat model. Biochemical analysis, HE staining, and PAS staining were used to assess the effects of bajijiasu on HN rat model. Western blotting was used to analyze the potential mechanisms. RESULTS: The results of HE staining and PAS staining showed that bajijiasu could alleviate the pathological changes in HN rat models; biochemical analysis found that the concentration of Malondialdehyde (MDA), total protein (TP), albumin (ALB), microalbuminuria (MALB), blood urea nitrogen (BUN), creatinine (Cr), triglyceride (TG), and low-density lipoprotein-cholesterol (LDL-C) were significantly decreased compared with the model group after bajijiasu treatment; and bajijiasu could regulate the expression of TNF-α, IL-6, MDA, SOD1 and AGEs in HN rats; the result of western blotting demonstrated that bajijiasu could down-regulate the expression of TGFß1, NOX4, JNK, p- JNK and up-regulate the expression PPARγ and SOD 1 in HN rats. CONCLUSION: Those results demonstrated that bajijiasu could alleviate the pathological changes and physiological and biochemical symptoms of HN rat models by regulating the expression of TGFß1, PPARγ, JNK, p-JNK, NOX4 and SOD1 but could not lower the blood pressure of HN rats. Those pieces of evidence may provide a new therapeutic method for HN treatment.

Biochanin A protects against iron overload associated knee osteoarthritis via regulating iron levels and NRF2/System xc-/GPX4 axis.

BACKGROUND: Iron homeostasis plays a positive role in articular cartilage health. Excessive iron or iron overload can induce oxidative stress damage in chondrocytes and ferroptosis cell death, advancing knee osteoarthritis (KOA). However, up to date, few effective agents treat iron overload-induced KOA (IOKOA). Chinese herbal medicine (CHM) provides abundant resources for drug selection to manage bone metabolic conditions, including osteoporosis. Biochanin A (BCA) is a novel bioactive multifunctional natural compound isolated from Huangqi, which has protective effects on bone loss. Nevertheless, the function and mechanism of BCA in treating IOKOA are still elusive. PURPOSE: This study seeks to uncover the potential therapeutic targets and mechanisms of BCA in the management of KOA with iron accumulation. METHODS: Iron dextrin (500 mg/kg) was intraperitoneally injected into mice to establish the iron overloaded mice model. OA was induced through surgery, and the progression was evaluated eight weeks following surgery. OA severity was evaluated with micro-CT and Safranin-O/Fast green staining in vivo. Iron deposition in the knee joint and synovium was assessed using Perl's Prussian blue staining. Ferric ammonium citrate (FAC) was then administered to primary chondrocytes to evaluate iron regulators mediated iron homeostasis. Toluidine blue staining was utilized to identify chondrocytes in vitro. The vitality of the cells was assessed using the CCK-8 test. The apoptosis rate of cells was measured using Annexin V-FITC/PI assay. The intracellular iron level was detected utilizing the calcein-AM test. Reactive oxygen species (ROS), lipid-ROS, and mitochondrial membrane potentiality were reflected via fluorescence density. Utilizing RT-qPCR and western blotting, the expression level was determined. RESULTS: Micro-CT and histological staining of knee joints showed greater cartilage degradation and higher iron buildup detected in iron-overloaded mice. BCA can reduce iron deposition and the severity of KOA. Toluidine blue staining and the CCK-8 assay indicated that BCA could rescue chondrocytes killed by iron. Cell apoptosis rates were increased due to iron overload but improved by BCA. Further, the intracellular content of iron, ROS, and lipid-ROS was increased with ferric ammonium citrate (FAC) treatment but restored after treatment with different concentrations of BCA. JC-1 staining revealed that BCA could reduce mitochondrial damage induced by iron overload. CONCLUSION: Iron overload was shown to promote chondrocyte ferroptosis in vivo and in vitro. Moreover, iron overload suppressed the expression of collagen II and induced MMP expression by catalyzing ROS generation with mitochondrial dysfunction. Our results showed that BCA could directly reduce intracellular iron concentration by inhibiting TfR1 and promoting FPN but also target the Nrf2/system xc-/GPX4 signaling pathway to scavenge free radicals and prevent lipid peroxidation. The results of this research indicate that BCA regulates iron homeostasis during the progression of osteoarthritis, which can open a new field of treatment for KOA.

Yishen Huashi Granules Ameliorated the Development of Diabetic Nephropathy by Reducing the Damage of Glomerular Filtration Barrier.

Background: Diabetic nephropathy (DN) is one of the most common complications of diabetes and the primary cause of end-stage renal disease. At present, renin-angiotensin-aldosterone system (RAAS) blockers have been applied as first-class drugs to restrain development of DN; however, its long-term effect is limited. Recent evidence has shown definite effects of Chinese medicine on DN. Yishen Huashi (YSHS) granule is a traditional Chinese Medicine prescription that has been used in the clinic to treat DN, but its mechanism is not understood. Methods: In the present study, both in vitro and in vivo studies were carried out. The DN model was induced by STZ in Wistar rats, and GEnC and HPC cell lines were applied in the in vitro study. Quality of YSHS was evaluated by LC-MS/MS. A metabolomic study of urine was carried out by LC-MS; influence of YSHS on composition of DN was analyzed by network pharmacology. Mechanism of the YSHS on DN was analyzed by Q-PCR, Western Blot, and multi-immunological methods. Results: We found YSHS administration significantly reduced levels of HbA1c and mALB. Histopathological analysis found that YSHS preserved integrity of glomerular filtration barrier by preserving viability of glomerular endothelial cells and podocytes, inhibiting glomerular fibrosis, reducing oxidative stress damage, and enhancing cross-talk among glomerular endothelial cells and podocytes. Network pharmacology, differential metabolite analysis, as well as intracellular pathway experimental study demonstrated that the PI3K/AKT/mTOR signaling pathway played a pivotal role in it. Conclusion: Our present findings supplied new understanding toward the mechanism of YSHS on inhibiting DN.

Naringenin protects against iron overload-induced osteoarthritis by suppressing oxidative stress.

BACKGROUND: The traditional Chinese medicine Gusuibu, the rhizome of Rhizoma Drynariae, is used to treat rheumatism and fractures. Naringenin (NAR) is an active ingredient in Gusuibu and has significant anti-inflammatory and antioxidant effects. However, the role of naringenin in iron overload-induced osteoarthritis (IOOA) is unknown. HYPOTHESIS: NAR reduces cartilage damage in IOOA. METHODS: The effects of NAR on the viability of IOOA chondrocytes and the synthesis ability of type II collagen were evaluated using cell counting kit (CCK8) and toluidine blue assays. To determine the mechanism of action and characteristics of NAR, the intracellular iron ion content, apoptosis rate, and mitochondrial membrane potential (MMP) change, and malondialdehyde (MDA) levels, as well as the degree of reactive oxygen species (ROS) and lipid hydroperoxide (LPO) accumulation in the cells were detected in vitro and verified using western blotting and quantitative real-time PCR (qRT-PCR). To verify the role of NAR in vivo, IOOA mice were established using iron dextran and surgery-induced destabilised medial meniscus. Changes in the articular cartilage and subchondral bone were examined using Safranin O-fast Green staining (S-O), haematoxylin-eosin staining (H&E), and microcomputed tomography (µCT). RESULTS: In vitro, NAR attenuated the impairment of cell viability, apoptosis, and MMP caused by ferric ammonium citrate and interleukin-1ß co-culture, increased the levels of MDA, reduced the expression of matrix metallopeptidase (MMP)3, MMP13, and Bax, and restored the expression of type II collagen (Col II). NAR showed a slight iron accumulation-reducing effect. NAR alleviated the accumulation of ROS and LPO in IOOA chondrocytes and upregulated antioxidant genes nuclear factor E2-related factor 2 (NRF2) and haem oxygenase 1 (HO-1). When ML385, a specific NRF-2 inhibitor, was added, the protective effect of NAR was significantly inhibited. In vivo, NAR reduced synovitis and attenuated cartilage damage and subchondral bone proliferation in IOOA mice. CONCLUSIONS: NAR can reduce oxidative stress through the NRF2-HO-1 pathway, alleviate cartilage damage under iron overload, and has the potential to treat IOOA.

Bioinformatics analysis combined with experimental validation to explore the mechanism of XianLing GuBao capsule against osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: XianLing GuBao Capsule (XLGB) is often used to treat osteoarthritis (OA), osteoporosis, fractures, and other musculoskeleton disorders. However, the molecular mechanism of XLGB for treating OA is still unclear. AIM OF THE STUDY: This study set out to uncover the molecular mechanism underlying the treatment of osteoarthritis with XLGB. MATERIALS AND METHODS: Disease genes were obtained from CTD, DisGeNET, and GeneCards databases, and XLGB drug targets were obtained from ETCM and target genes predicted by XLGB metabolic components reported in the literature. Then we used the Venn diagram viewer to extract disease and drug intersection genes as potential therapeutic genes for Protein-protein interaction (PPI), GO terminology, and KEGG pathway analysis. Subsequently, we performed qRT-PCR, Western blot and histological analysis to validate the therapeutic effect of XLGB against OA and its molecular mechanism. RESULTS: A total of 1039 OA genes and 949 XLGB target genes were collected, and finally 188 potential therapeutic target genes were obtained. PPI network analysis indicated that the main target genes for XLGB to treat OA include Akt1, Mapk3, Il-6, Il-1ß, Ptgs2, Mmp9, etc. The results of KEGG and GO enrichment analysis suggested that XLGB may treat OA by anti-inflammatory and reducing extracellular matrix degradation. In vitro, XLGB down-regulated the expressions of Mmp3, Mmp9, Mmp12, Mmp13, Cox-2, Il-6, increased the expression of Collagen II and Sox9. Mechanistically, XLGB inhibits the activation of PI3K/AKT/NF-κB and MAPK pathways. Moreover, the results of animal experiments indicated that XLGB reduced cartilage destruction, bone resorption, and synovitis in osteoarthritic rats. CONCLUSIONS: XLGB has a protective effect against OA by suppressing PI3K/AKT/NF-κB and MAPK signaling. Our study provides a theoretical basis for XLGB in the treatment of osteoarthritis.

Gegen Qinlian Decoction ameliorates type 2 diabetes osteoporosis via IGFBP3/MAPK/NFATc1 signaling pathway based on cytokine antibody array.

BACKGROUND: Osteoporosis affects more than half the patients with type 2 diabetes mellitus (T2DM). Up to data, there is no effective clinical practice in managing type 2 diabetes osteoporosis (T2DOP) because of its complex pathogenesis. Gegen Qinlian Decoction (GQD) has been used for the long-term management of T2DM. However, the underlying mechanism of GQD in the treatment of T2DOP remains unknown. PURPOSE: To reveal the role of GQD in T2DOP and its potential therapeutic targets in the management of T2DOP. STUDY DESIGN: The effect of GQD on T2DOP was observed in db/db mice in four groups: model group, GQD low-dose group (GQD-L), GQD high-dose group (GQD-H), and metformin (positive control) group. C57BL/6J mice were used as the negative control group. METHODS: Quantitative phytochemical analysis of GQD was performed using high-performance liquid chromatography (HPLC). Micro-CT and hematoxylin-eosin (H&E) staining were used to evaluate bone histomorphometry. To screen for candidate targets of GQD, a cytokine antibody array was used, followed by bioinformatics analysis. Quantitative real-time PCR (qRT-PCR) and western blotting (WB) were used to determine expression levels. RESULTS: The major active components of GQD were confirmed by HPLC. Micro-CT and H&E staining showed that bone mass was significantly increased in the GQD-H group compared with the model group. Antibody arrays revealed that the expression of insulin-like growth factor binding protein 3 (IGFBP3) was elevated in the GQD-H group. The MAPK pathway was identified using bioinformatics analysis. Additionally, the levels of osteoclastogenesis-related genes, including cathepsin K (Ctsk), acid phosphatase 5 (Acp5), matrix metallopeptidase 9 (Mmp9), and ATPase H+ transporting V0 subunit D2 (Atp6v0d2) were significantly decreased in the GQD-H group. Compared with the model group, high-dosage GQD inhibited phosphorylation of extracellular signal-regulated kinases (ERKs) and P38 mitogen-activated protein kinase (MAPK) and the expression of c-Fos and nuclear factor of activated T cells 1 (NFATc1). CONCLUSION: GQD plays a protective role in T2DOP by upregulating IGFBP3 expression and downregulating the IGFBP3/MAPK/NFATc1 signaling pathway. IGFBP3 in serum may also be a novel biomarker in the treatment of T2DOP. Our current findings not only expand the application of GQD, but also provide a theoretical basis and guidance for T2DOP.

12-Deoxyphorbol 13-acetate inhibits RANKL-induced osteoclastogenesis via the attenuation of MAPK signaling and NFATc1 activation.

Osteoporosis, characterized by bone loss and microstructure damage, occurs when osteoclast activity outstrips osteoblast activity. Natural compounds with inhibitory effect on osteoclast differentiation and function have been evidenced to protect from osteoporosis. After multiple compounds screening, 12-deoxyphorbol 13-acetate (DPA) was found to decline RANKL-induced osteoclastogenesis dose-dependently by attenuating activities of NFATc1 and c-Fos, followed by decreasing the level of osteoclast function-associated genes and proteins including Acp5, V-ATPase-d2 and CTSK. Mechanistically, we found that DPA suppressing RANKL-induced downstream signaling pathways, including MAPK signaling pathway and calcium oscillations. Furthermore, the in vivo efficacy of DPA was further confirmed in an OVX-induced osteoporosis mice model. Collectively, the results in our presentation reveal that DPA might be a promising compound to manage osteoporosis.

Sanhuang Jiangtang tablet protects type 2 diabetes osteoporosis via AKT-GSK3ß-NFATc1 signaling pathway by integrating bioinformatics analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Sanhuang Jiangtang tablet (SHJTT), has been widely used to treat type 2 diabetes mellitus (T2DM). However, the potential and mechanism of SHJTT in treating type 2 diabetes osteoporosis (T2DOP) has not been reported. AIM OF THE STUDY: The aim of this work was to investigate the role and the underlying molecular mechanism of SHJTT in managing type 2 diabetes osteoporosis. MATERIALS AND METHODS: The target genes of each component consisting of SHJTT were obtained by searching the ETCM database. The target genes of osteoporosis and diabetes were individually acquired by analyzing the DisGeNET and OMIM disease databases. Then the potential therapeutic genes were obtained from the intersection of the herbal medicine targets and the disease targets which were imported into the R and STRING platform for the analysis of GO terms, KEGG pathways and PPI network. The key modules of PPI network were constructed by Cytoscape software. Finally, leptin receptor deficiency (db/db) mice were confirmed as an animal model of type 2 diabetic osteoporosis (T2DOP) through phenotype assessment and the key genes of SHJTT against T2DOP were validated by quantitative real-time PCR (qRT-PCR). RESULTS: A total of 786 target genes of SHJTT were obtained from ETCM. Simultaneously, a total of 3906 osteoporosis and type 2 diabetes associated targets were acquired from DisGeNET and OMIM databases. Then, 97 common targets were found by overlapping them. On the basis of the GO and KEGG enrichment analysis and PPI network, we found that the related pathway of SHJTT in type 2 diabetes osteoporosis was AKT-GSK3ß-NFATc1 pathway which is tightly associated with osteoclast differentiation. The expression of key genes including Akt1, Mapk3, Gsk3ß, Mmp9, Nfkb1 were significantly down-regulated by SHJTT in T2DOP mice (p < 0.05). CONCLUSIONS: SHJTT had a protective effect on T2DOP via regulating AKT-GSK3ß-NFATc1 signaling pathway. This study might provide a theoretical basis for the application of SHJTT for the treatment of type 2 diabetic osteoporosis.