Tanshinol alleviates impaired bone formation by inhibiting adipogenesis via KLF15/PPARγ2 signaling in GIO rats.

Glucocorticoid (GC)-induced osteoporosis (GIO) is characterized by impaired bone formation, which can be alleviated by tanshinol, an aqueous polyphenol isolated from Salvia miltiorrhiza Bunge. In this study we investigated the molecular mechanisms underlying GC-induced modulation of osteogenesis as well as the possibility of using tanshinol to interfere with GIO. Female SD rats aged 4 months were orally administered distilled water (Con), prednisone (GC, 5 mg·kg-1·d-1), GC plus tanshinol (Tan, 16 mg·kg-1·d-1) or GC plus resveratrol (Res, 5 mg·kg-1·d-1) for 14 weeks. After the rats were sacrificed, samples of bone tissues were collected. The changes in bone formation were assessed using Micro-CT, histomorphometry, and biomechanical assays. Expression of Kruppel-like factor 15 (KLF15), peroxisome proliferator-activated receptor γ 2 (PPARγ 2) and other signaling proteins in skeletal tissue was measured with Western blotting and quantitative RT-PCR. GC treatment markedly increased the expression of KLF15, PPARγ2, C/EBPα and aP2, which were related to adipogenesis, upregulated FoxO3a pathway proteins (FoxO3a and Gadd45a), and suppressed the canonical Wnt signaling (ß-catenin and Axin2), which was required for osteogenesis. Thus, GC significantly decreased bone mass and bone quality. Co-treatment with Tan or Res effectively counteracted GC-impaired bone formation, suppressed GC-induced adipogenesis, and restored abnormal expression of the signaling molecules in GIO rats. We conclude that tanshinol counteracts GC-decreased bone formation by inhibiting marrow adiposity via the KLF15/PPARγ2/FoxO3a/Wnt pathway.

Supplementation of ketoacids contributes to the up-regulation of the Wnt7a/Akt/p70S6K pathway and the down-regulation of apoptotic and ubiquitin-proteasome systems in the muscle of 5/6 nephrectomised rats.

Ketoacids (KA) are known to improve muscle mass among patients with chronic kidney disease (CKD) on a low-protein diet (CKD-LPD), but the mechanism of its preventive effects on muscle atrophy still remains unclear. Since muscle atrophy in CKD may be attributable to the down-regulation of the Wnt7a/Akt/p70S6K pathway and the activation of the ubiquitin-proteasome system (UPS) and the apoptotic signalling pathway, a hypothesis can readily be drawn that KA supplementation improves muscle mass by up-regulating the Wnt7a/Akt/p70S6K pathway and counteracting the activation of the UPS and caspase-3-dependent apoptosis in the muscle of CKD-LPD rats. Rats with 5/6 nephrectomy were randomly divided into three groups, and fed with either 22 % protein (normal-protein diet; NPD), 6 % protein (LPD) or 5 % protein plus 1 % KA for 24 weeks. Sham-operated rats with NPD intake were used as the control. The results demonstrated that KA supplementation improved protein synthesis and increased related mediators such as Wnt7a, phosphorylated Akt and p70S6K in the muscle of CKD-LPD rats. It also inhibited protein degradation, withheld the increase in ubiquitin and its ligases MAFbx (muscle atrophy F-box) and MuRF1 (muscle ring finger-1) as well as attenuated proteasome activity in the muscle of CKD-LPD rats. Moreover, KA supplementation gave rise to a reduction in DNA fragment, cleaved caspase-3 and 14 kDa actin fragment via the down-regulation of the Bax:Bcl-2 ratio in the muscle of CKD-LPD rats. The beneficial effects unveiled herein further consolidate that KA may be a better therapeutic strategy for muscle atrophy in CKD-LPD.

Tubson-2 decoction ameliorates rheumatoid arthritis complicated with osteoporosis in CIA rats involving isochlorogenic acid A regulating IL-17/MAPK pathway.

BACKGROUND: Osteoporosis (OP) is considered as one of the major comorbidities of rheumatoid arthritis (RA), and is responsible for fragility fracture. However, there is currently no effective treatment for RA complicated with OP. Tubson-2 decoction (TBD), a Mongolian medicine also known as Erwei Duzhong Decoction, has been shown to exert a preventive effect on post-menopausal osteoporosis (PMOP). The preventive effects of TBD on RA-induced OP, as well as the bioactive compound responsible and the underlying mechanisms, remain to be elucidated. OBJECTIVE: To explore the effects of TBD on RA-induced OP in vivo, and to elucidate the mechanism of isochlorogenic acid A (ICA), the effective component of TBD, in vitro. METHODS: To evaluate the anti-arthritic and anti-osteoporotic effects of TBD, we conducted H&E straining and safranine O/fast green, TEM, immunohistochemistry (IHC), bone histomorphometry, micro-CT imaging, and biomechanical testing in collagen induced arthritis (CIA) rats. The active ingredient in TBD was identified using network pharmacology and molecular docking. The identification was supported by in vivo IHC assay, and further confirmed using qRT-PCR, Western blot, and SEM analysis in TNF-α-treated MH7A cells and/or in LPS-exposed RAW264.7 cells. RESULTS: Oral administration of TBD attenuated the severity of arthritis and osteopenia as well as poor bone quality, in CIA rats. Additionally, TBD and the positive control, tripterygium glycosides (TG), exhibited similar effects in reducing inflammation in both the synovium and ankle joint. They also were both effective in improving bone loss, microarchitecture, and overall bone quality. TBD reduced the expression of MMP13, IL-17, and p-JNK protein in the synovium of CIA rats. ICA, which was screened, suppressed TNF-α or LPS-triggered inflammatory responses via down-regulating IL-17 signaling, involving in MMP13, IL-1ß, IL-23, and IL-17, and the MAPK pathway including p-ERK, p-JNK, and p-P38, both in MH7A cells and in RAW264.7 cells. Furthermore, ICA prevented osteoclasts from differentiating and bone resoprtion in a dose-dependent manner in vitro. CONCLUSION: This study provides the first evidence that TBD exerts intervening effects on RA-induced OP, possibly through the downregulation of the IL-17/MAPK signaling pathway by ICA. The findings of our study provides valuable insights for further research in this area.

Overexpression of ABCB1 confers resistance to FLT3 inhibitor FN-1501 in cancer cells: in vitro and in vivo characterization.

FN-1501 is a potent FLT3 inhibitor with antitumor activity. A phase 1 trial of FN-1501 monotherapy in patients with advanced solid tumors and R/R AML is in progress. Since one of the primary causes of multidrug resistance (MDR) is the overexpression of ATP-binding cassette superfamily B member 1 (ABCB1), the objective of this study was to investigate the potential relationship between FN-1501 and the ABCB1 transporter. We found ABCB1 overexpressing-cancer cells conferred FN-1501 resistance, which could be reversed by an ABCB1 inhibitor. Molecular docking study revealed that FN-1501 docked the ligand binding site with an affinity score of -9.77 kcal/mol, denoting a strong interaction between FN-1501 and ABCB1. Additionally, the ABCB1 ATPase assay indicated that FN-1501 could significantly stimulate ABCB1 ATPase activity. Furthermore, we observed a similar trend of ABCB1-facilated FN-1501 resistance in tumor-bearing mice model. In sum, we demonstrate that FN-1501 is a substrate of ABCB1 transporter from both in vivo and in vitro studies. Therefore, our findings provide new insight on the mechanism of chemoresistance due to ABCB1 overexpression.

Overexpression of ABCB1 Associated With the Resistance to the KRAS-G12C Specific Inhibitor ARS-1620 in Cancer Cells.

The KRAS-G12C inhibitor ARS-1620, is a novel specific covalent inhibitor of KRAS-G12C, possessing a strong targeting inhibitory effect on KRAS-G12C mutant tumors. Overexpression of ATP-binding cassette super-family B member 1 (ABCB1/P-gp) is one of the pivotal factors contributing to multidrug resistance (MDR), and its association with KRAS mutations has been extensively studied. However, the investigations about the connection between the inhibitors of mutant KRAS and the level of ABC transporters are still missing. In this study, we investigated the potential drug resistance mechanism of ARS-1620 associated with ABCB1. The desensitization effect of ARS-1620 was remarkably intensified in both drug-induced ABCB1-overexpressing cancer cells and ABCB1-transfected cells as confirmed by cell viability assay results. This desensitization of ARS-1620 could be completely reversed when co-treated with an ABCB1 reversal agent. In mechanism-based studies, [3H] -paclitaxel accumulation assay revealed that ARS-1620 could be competitively pumped out by ABCB1. Additionally, it was found that ARS-1620 remarkably stimulated ATPase activity of ABCB1, and the HPLC drug accumulation assay displayed that ARS-1620 was actively transported out of ABCB1-overexpressing cancer cells. ARS-1620 acquired a high docking score in computer molecular docking analysis, implying ARS-1620 could intensely interact with ABCB1 transporters. Taken all together, these data indicated that ARS-1620 is a substrate for ABCB1, and the potential influence of ARS-1620-related cancer therapy on ABCB1-overexpressing cancer cells should be considered in future clinical applications.

Role of the Wnt/ß-catenin signaling pathway in inducing apoptosis and renal fibrosis in 5/6-nephrectomized rats.

Renal fibrosis is the final common pathway of all progressive renal disease. Excessive and chronic activation of the Wnt/ß-catenin signaling pathway results in chronic kidney disease (CKD) progression. To mimic CKD, the present study used 5/6-nephrectomized rats, and alterations in kidney histology, expression of ß­catenin and renal cell apoptosis were assessed. In addition, mesangial cells were cultured in vitro and transfected with ß­catenin siRNA to evaluate the effect of blocking Wnt/ß­catenin signaling on cell apoptosis and the expression of markers of renal fibrosis. The results demonstrated that CKD rat kidney tissues exhibited moderate renal fibrosis and significantly increased expression levels of ß­catenin and apoptosis associated proteins compared with sham­operated rats. In vitro, silencing of ß-catenin by siRNA attenuated tumor necrosis factor­α­induced apoptosis and decreased mRNA expression levels of various markers of fibrosis, including fibronectin, transforming growth factor­ß, and collagen I, III and IV. In conclusion, inhibition of Wnt/ß­catenin signaling by ß­catenin silencing attenuated apoptosis and expression of fibrosis-associated markers in renal cells. The present study suggested that the Wnt/ß­catenin signaling pathway may serve as a potential treatment strategy for renal fibrotic disorders.

Effect of HDAC-6 on PD cell induced by lactacystin.

OBJECTIVE: To explore the effects of histone deacetylase 6(HDAC-6) on the PD cell model induced by proteasome inhibitor lactacystin. METHODS: Human neuroblastoma SK-N-SH cells were cultured. The wild type pcDNA3.1-alpha-synuclein eukaryotic expression plasmid was transferred into the cells which then were divided into control group, group L, group T and group T+L. The cells of group L were added with 5 µmol/L lactacystin dissolved indimethylsulfoxide (DMSO) to induce PD cell model with abnormal protein aggregation, the cells of control group were treated with 5 µmol/L DMSO, the cells of group T were treated with 5 µmol/L selective HDAC-6 inhibitor tubacin dissolved in DMSO, and the cells of group T+L were treated with 5 µmol/L lactacystin and 10 µmol/L tubacin dissolved in DMSO. The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 were detected by Western blot and the cell survival rate of all the groups was detected by MTT colorimetric assay, and compared 24 h after the cells were treated. RESULTS: The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 of the cells of group L were significantly higher than the control group, and the cell survival rate was significantly lower (P < 0.05); the expression level of alpha-synuclein oligomers of the cells of group T+L was significantly higher than group L, but the expression level of HSP-27 and HSP-70 were significantly lower, and so as the cell survival rate (P < 0.05); the differences of the expression level of alpha-synuclein oligomers, HSP-27 and HSP-70 and the cell survival rate of the cells of group T and the control group were not statistically significant (P > 0.05). CONCLUSIONS: The expression level of alpha-synuclein oligomers can be improved and the cell survival rate can be reduced by the PD cell model induced by lactacystin and treated with selective HDAC-6 inhibitor tubacin, which means that alpha-synuclein oligomers of the PD cell model induced by lactacystin can be inhibited and the cell survival rate can be improved by HDAC-6, and the mechanism may be related to the increased of HSP-27 and HSP-70.

Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease.

Our evidence demonstrated that CKD upregulated the expression of myostatin, TNF-α, and p-IkBa and downregulated the phosphorylation of PI3K, Akt, and FoxO3a, which were also associated with protein degradation and muscle atrophy. The autophagosome formation and protein expression of autophagy-related genes were increased in muscle of CKD rats. The mRNA level and protein expression of MAFbx and MuRF-1 were also upregulated in CKD rats, as well as proteasome activity of 26S. Moreover, activation of myostatin elicited by TNF-α induces C2C12 myotube atrophy via upregulating the expression of autophagy-related genes, including MAFbx and MuRF1 and proteasome subunits. Inactivation of FoxO3a triggered by PI3K inhibitor LY294002 prevented the myostatin-induced increase of expression of MuRF1, MAFbx, and LC3-II protein in C2C12 myotubes. The findings were further consolidated by using siRNA interference and overexpression of myostatin. Additionally, expression of myostatin was activated by TNF-α via a NF-κB dependent pathway in C2C12 myotubes, while inhibition of NF-κB activity suppressed myostatin and improved myotube atrophy. Collectively, myostatin mediated CKD-induced muscle catabolism via coordinate activation of the autophagy and the ubiquitin-proteasome systems.

Tanshinone IIA attenuates renal fibrosis and inflammation via altering expression of TGF-ß/Smad and NF-κB signaling pathway in 5/6 nephrectomized rats.

PURPOSE: In traditional Chinese medicine, Tanshinone IIA is used to treat chronic kidney disease (CKD). However, its biological activity and mechanism of action in renal fibrosis and inflammation are not fully identified. The current study was conducted to determine the effects of Tanshinone IIA treatment on CKD by assessing potential modulation of the TGF-ß/Smad and NF-κB signaling pathway. METHODS: CKD was produced in rats by 5/6 nephrectomy. They were then divided into the following groups: control (sham operation); CKD (5/6 nephrectomy); 5/6 nephrectomy+Tanshinone IIA (10mg/kg in average, once a day for 16 weeks). Serum and urine samples were obtained from animals in each group, and serum creatinine (Scr), blood urea nitrogen (BUN) levels and 24h urinary protein excretion were measured. Tissue samples from the kidney were used for morphometric studies (Masson's trichrome). The expression of fibronectin protein and collagen types I, III, IV, and TGF-ß, TNF-α, CXCL-1, MCP-1, RANTES mRNA were evaluated using immunohistochemistry and RT-PCR analysis; the TGF-ß/Smad and NF-κB signaling pathway was detected by immunohistochemistry and Western blot analysis. RESULTS: The following effects were observed in CKD rats treated with Tanshinone IIA: (1) marked improvements in Scr, and 24h urine protein excretion; (2) significant reductions in protein and mRNA levels of fibronectin, collagen III, and collagen IV and TNF-α, MCP-1, and CXCL-1; (3) significantly inhibited the TGF-ß/Smad and NF-κB signaling activation. CONCLUSIONS: These results suggest that Tanshinone IIA suppresses renal fibrosis and inflammation via altering expression of TGF-ß/Smad and NF-κB pathway in the remnant kidney, thus supporting the potential of Tanshinone IIA as a new therapeutic agent for slowing the progression of CKD.

Resveratrol prevents TNF-α-induced muscle atrophy via regulation of Akt/mTOR/FoxO1 signaling in C2C12 myotubes.

Muscle atrophy poses a serious concern to patients inflicted with inflammatory diseases. There is now increasing evidence which suggests a vital role for tumor necrosis factor alpha (TNF-α) in muscle pathology associated with impairment of differentiation and muscle wasting. Resveratrol has been an ascribed inhibitory effect on glucocorticoid-induced muscle atrophy in vitro, but the influence of resveratrol on the growth of C2C12 myotubes exposed to TNF-α remains unclear. The present study aimed to investigate the involvement of TNF-α in the regulation of skeletal muscle hypertrophy and atrophy, and the possibility to interfere with such modulations by means of resveratrol supplementation. For this purpose, C2C12 myotubes were treated with TNF-α in the presence or absence of resveratrol. Myotube treatment with TNF-α contributes to both hyperexpression of the muscle-specific ubiquitin ligase MAFbx and MuRF1, and these alterations are linked to a decrease of anabolic targets (Akt, mTOR, p70S6k and 4E-BP1) and an increase of catabolic targets (FoxO1, FoxO3a, MAFbx and MuRF1). Resveratrol supplementation effectively counteracts TNF-α induced muscle protein loss and reverses declining expression of Akt, mTOR, p70S6K, 4E-BP1and FoxO1, but exerts no influence of FoxO3a expression. Our study demonstrates that resveratrol can reverse the muscle cell atrophy caused by TNF-α through regulation of the Akt/mTOR/FoxO1 signaling pathways, followed by inhibition of the atrophy-related ubiquitin ligase. Our findings suggested that resveratrol could represent a possible strategy to improve muscle mass.

The p.R229Q variant of the NPHS2 (podocin) gene in focal segmental glomerulosclerosis and steroid-resistant nephrotic syndrome: a meta-analysis.

While many previous studies have reported an association between the p.R229Q variant of the NPHS2 gene and focal segmental glomerulosclerosis (FSGS) or steroid-resistant nephrotic syndrome (SRNS), a conclusive relationship has not been defined. In this study, we performed a meta-analysis of the published data to investigate the impact of the p.R229Q polymorphism on FSGS and SRNS patients. Despite significant heterogeneity within some of the comparisons, the results revealed significantly higher risks of SRNS in individuals homozygous for the variant allele (OR 7.411, 95% confidence interval 1.876-29.436, p = 0.004) compared to homozygous non-variant individuals. However, the carrier rate of the p.R229Q variant was not significantly different between SRNS patients and steroid-sensitive nephrotic syndrome patients. No statistically significant differences in the p.R229Q carrier rate were observed between FSGS patients and controls or FSGS patients and patients with different pathology classifications. No notable differences in the p.R229Q carrier rate were found between patients and controls in any group with early-onset disease (onset age < 18). In conclusion, our meta-analysis suggests that for adult-onset disease (onset age > 18), the homozygous variant could be a potential predictor of hereditary nephrotic syndrome and that the p.R229Q allele cannot currently be considered a risk factor for predicting FSGS.

Astragalus polysaccharide improves muscle atrophy from dexamethasone- and peroxide-induced injury in vitro.

Astragalus polysaccharide (APS) is an important bioactive component of Astragalus membranaceus Bunge (Leguminosae) that has been used in traditional Chinese medicine for treating muscle wasting, a serious complication with complex mechanism manifested as myofibers atrophy and satellite cells apoptosis. In this study, the anti-atrophy and anti-apoptotic activity of Astragalus polysaccharide (APS) was characterized in C2C12 skeletal muscle myotubes and myoblasts. APS inhibited dexamethasone-induced atrophy by restoring phosphorylation of Akt, m-TOR, P70s6k, rpS6 and FoxO3A/FoxO1. The targets that protected C2C12 myoblasts from damage by H2O2 were promoting cells proliferation and inhibiting cells apoptosis. The protective mechanisms involved mitochondrial pathway and death receptor pathway. Moreover, Antioxidant effect of APS was also detected in this work. Our findings suggested that APS could be explored as a protective and perhaps as a therapeutic agent in the management of muscle wasting.