Effect of Erxian Decoction-containing serum on H_2O_2-induced proliferation and osteogenic differentiation of MC3T3-E1 cells via BK channels / 中国中药杂志

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.

Research progress of miRNAs in exosomes derived from bone-related cells regulating bone formation / 中国药理学通报

Exosome is a goblet-shaped extracellular vesicle with a lipid bilayer structure, and its diameter is about 30-150 nm. Exosomes participate in cell-to-cell communication by transporting proteins, miRNAs, and other biologically active factors, thereby affecting recipient cells. In recent years, a lot of studies have shown that exosomes derived from bone-related cells can not only mediate intercellular communication within the bone marrow microenvironment in a range of physiological conditions, but also contribute to the multiple pathological conditions in skeletal disorders. Here, we summarized the regulation of miR-NA in exosomes derived from bone-related cells on bone formation.

Effect of Erxian Decoction on proteomics and PI3K signaling pathway of osteoblasts stimulated by H2O2 / 中国中药杂志

The present study aimed to explore the effect of Erxian Decoction on proteomics of osteoblasts stimulated by hydrogen peroxide(H_2O_2) and its protective mechanism with the H_2O_2-induced cell model of oxidative stress. The primary osteoblasts were cultured from the skulls of newborn rats(within 24 hours) and divided into a control group, a model group, a Fosamax group, and an Erxian Decoction group. Blank serum was added in the control group and model group, and the drug-containing serum was added correspondingly to the remaining two groups. After 45 hours, H_2O_(2 )stimulation was conducted for three hours except for the control group, followed by protein extraction. Nano-LC-LTQ-Orbitrap system was used for protein detection, Protein Discovery for protein identification, and SIEVE for quantitative and qualitative analysis. Furthermore, following the blocking of PI3 K signaling pathway by LY294002(10 μmol·L~(-1)), a control group, a model group, an LY294002 group, an Erxian Decoction group, and an Erxian Decoction + LY294002 group were set up to observe the effect of Erxian Decoction on cell proliferation, alkaline phosphatase(ALP) activity, and the relative expression of BMP-2, OPG, p-Akt, p-FoxO1 of osteoblasts stimulated by H_2O_2 under LY294002 intervention. The results revealed that 78 differential proteins were discovered between the Erxian Decoction group and model group, which were involved in the regulation of PI3 K/Akt, glucagon, estrogen, insulin, and other signaling pathways. LY294002 blunted the promoting effect of Erxian Decoction on osteoblast proliferation and significantly down-regulated the expression of OPG and p-FoxO1, whereas its down-regulation on the expression of BMP-2 and p-Akt was not significant. Both LY294002 and Erxian Decoction increased the ALP activity of osteoblasts, which may be related to the cell state and the cell differentiation. The above results suggest that Erxian Decoction can protect osteoblasts stimulated by H_2O_2, with the PI3 K/Akt signaling pathway as one of the internal mechanisms.

Effect of Gancao Fuzi Decoction on osteoarthritis and proteomics of articular cartilage in rats / 中国中药杂志

The rat osteoarthritis model was replicated by injection of sodium iodoacetate into the knee joint cavity, and the effects of Gancao Fuzi Decoction on rat osteoarthritis and the proteome of articular cartilage were investigated. Sixty SD rats weighing 230-250 g were randomly divided into normal group, model group, glucosamine sulfate group, and Gancao Fuzi Decoction high, medium and low dose groups. Osteoarthritis model was induced by intra-articular injection of sodium iodoacetate(3 mg on each leg) in all groups except the normal group. After modeling, each administration group was given intragastric administration for 1 month. During the administration period, joint pain test and joint width measurement were performed every week to observe the autonomous behavior of rats. Enzyme linked immunosorbent assay(ELISA) method was used to detect the contents of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), matrix metalloproteinase-3(MMP-3), and matrix metalloproteinase tissue inhibitor(TIMP-1) in rat joint lavage fluid. Hematoxylin-eosin(HE) staining was used to observe bone and joint morphology. Nano-LC-LTQ-Orbitrap system was used to detect arti-cular cartilage proteins. The results showed that, compared with the model group, Gancao Fuzi Decoction could significantly improve joint pain and joint swelling in osteoarthritis rats, significantly reduce the contents of TNF-α, IL-1β and MMP-3 in the joint cavity la-vage fluid, increase the content of TIMP-1, and relieve inflammatory diseases such as enlarged joint space, rough cartilage edge, different thickness of cartilage layer, and disordered arrangement of chondrocytes. After comparing the proteins between the groups, 273 differential proteins were screened out. KEGG analysis found that the above differential proteins involved 43 signaling pathways such as systemic lupus erythematosus, among which 11 signaling pathways were related to osteoarthritis. The above results indicated that Gancao Fuzi Decoction had a preventive effect on osteoarthritis, and its mechanism of action may be accomplished by regulating the protein expression of osteoarthritis-related signal pathways.

Effect of calcium on ion contents and expression of photosynthetic related genes in honeysuckle under salt stress / 中国中药杂志

Exogenous calcium can enhance the resistance of certain plants to abiotic stress. Research have demonstrated that exogenous calcium could enhances the resistance of honeysuckle under salt stress by promoting the transmission of photosynthetic electrons.The aim of this study was to investigate the effects of exogenous calcium on the contents of Na~+,K~+,Ca~(2+),Mg~(2+)and the expression of photosynthetic related genes Cab and rbc L. In this study,we used ICP-OES to analysis ion contents and used qRT-PCR to analysis the expression patterns of Cab and rbc L. The results showed that CaCl_2 significantly enhanced the K~+-Na~+,Ca~(2+)-Na~+,Mg~(2+)-Na+ratio of honeysuckle treated with 50 and 100 mmol·L~(-1) NaCl. Meanwhile,Cab and rbc L were significantly up-regulated under short-term salt stress,and CaCl_2 promoted this trend. From the two gene expression patterns,rbc L rapidly up-regulated on the first day of stress and then decreased,and was more sensitive to environmental changes. In summary,exogenous calcium could alleviate salt stress and increase plant development by increasing intracellular K~+-Na~+,Ca~(2+)-Na~+,Mg~(2+)-Na+ratio,and the transient overexpression of Cab and rbc L.