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.

Autophagy plays a protective role in cell death of osteoblasts exposure to lead chloride.

Lead (Pb) is a toxic heavy metal widespreadly used in industrial field. Prior studies showed that Pb exposure had detrimental effects on osteoblasts. The mechanisms underlying Pb-induced damage are complex. Autophagy can protect cells from various cytotoxic stimuli. In the present study, the aim of our research was to investigate whether Pb could activate autophagy to play a protective role against osteoblasts apoptosis. Our results indicated that PbCl2 induced autophagy and autophagic flux in MC3T3-E1 murine osteoblastic cell by RT-PCR, western blot, as well as fluorescence microscopy analysis of GFP-LC3, AO and MDC staining. Pb increased the apoptosis of osteoblasts, evidenced by western blot and Hoechst 33258 staining assessment. In addition, inhibiting autophagy by 3-MA further increased the osteoblasts apoptosis after Pb exposure, showed by flow cytometry and Hoechst 33258 staining. Furthermore, phosphorylation of mTOR and p70S6K was inhibited by Pb exposure, indicating that Pb might induce autophagy in osteoblasts via inhibiting mTOR pathway. Altogether, these evidence suggested that Pb exporsure promoted autophagy flux in osteoblasts. The activation of autophagy by Pb played a protective role in osteoblasts apoptosis, which might be mediated through the mTOR pathway.

Effect of 1,25-dihydroxyvitamin D3on regulatory T cells in ovariectomized mice.

OBJECTIVE: To investigate the correlation between regulatory T (Treg) cells and postmenopausal osteoporosis and the antiosteoporotic effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in relation to Treg cells. METHODS: Fifty female BALB/c mice were randomly divided into five groups: the basal control (BAS), Sham, ovariectomy (OVX), OVX+diethylstilbestrol (OVX+DES), and OVX+1,25(OH)2D3. Tibias were harvested and processed with decalcification for quantitative bone histomorphometry. Femurs were stained by immunohistochemistry to detect Foxp3 protein expression. Spleens were used to detect Treg and Foxp3 gene expression by flow cytometry and quantitative RT-PCR, respectively. RESULTS: In comparison with the Sham group, a significant decrease was found in the OVX group in such indices as trabecular bone volume/total tissue area (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th). 1,25(OH)2D3and DES partly prevented the decrease in BV/TV, Tb.N, Tb.Th in OVX mice. Treg cell number, Foxp3 mRNA expression in spleen and Foxp3 protein expression in femur significantly decreased in the OVX-treated group compared with those in the sham group. 1,25(OH)2D3and DES significantly increased Treg cell number and Foxp3 expression. Treg cells and Foxp3 gene expression were related to bone histomorphometric parameters. CONCLUSION: The decrease in Treg cell numbers is relevant to the postmenopausal osteoporosis. The antiosteoporosis of 1,25(OH)2D3is related to regulatory T cells.

Captopril improves osteopenia in ovariectomized rats and promotes bone formation in osteoblasts.

The present study was designed to investigate the effects of captopril, an angiotensin-converting enzyme inhibitor (ACEI), on bone loss in aged ovariectomized (OVX) rats and its impact on the differentiation of cultured primary osteoblasts. Ten-month-old female Sprague-Dawley rats were used for the study. After 2 months post ovariectomy (OVX), the rats were treated with captopril (1 or 5 mg/kg/day, respectively) for another 2 months. At endpoint, trabecular bone of the fourth lumbar vertebrae (L4) was undecalcified and examined by bone histomorphometry; the fifth lumbar vertebrae (L5) were examined by compression test. Primary osteoblasts were isolated from the calvaria of newborn rats and treated with different concentrations of captopril in a different durations. The content of secreted alkaline phosphatase (ALP) and mRNA expression of collagen I in osteoblasts were determined to demonstrate osteoblast bone formation. In aged rats with estrogen deficiency-induced osteopenia, captopril increased the trabecular area (%BV/TV) of L4 up to 33% and improved biomechanical properties by increasing L5 break stress and elastic modulus when compared to those in the OVX group (P < 0.01). Captopril showed dose-dependent effects on promoting the secretion of ALP and increased mRNA expression of collagen I in the cultured rat osteoblasts. In summary, captopril, one of the most widely used ACEIs, has the potential effects of improving lumbar vertebral bone strength in aged OVX rats and promoting osteoblast bone formation in vitro.

[Synthetical evaluation of promoting effect of some kinds of transdermal enhancers with grey relational cluster method].

OBJECTIVE: Synthetical evaluation of promoting effect of some kinds of transdermal enhancers was carried through. METHOD: Diclofenac sodium was used as model, and azone and l-menthol and synthetic borneol and olieic acid and essential oil from Cnidium monnieri were used as transdermal enhancers. Transdermal absorption experimentation of diclofenac sodium on the device of penetrating skins in vitro was done. Cumulation of permeation amount and penetrating rates and steady fluxes and lag times were observed, and grey relational cluster method was used to evaluate the promoting effect of some kinds of transdermal enhancers. RESULT: As for promoting effect on diclofenac sodium, azone and l-menthol were the best, and synthetic borneol and olieic acid ranked behind. CONCLUSION: Grey relational cluster method can evaluate promoting effect objectively and fairly.