| ObjectiveDiscussion Jian Pi Formula (JPF) in vitro regulation of rat bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) osteogenic and adipogenic differentiation mechanism for the treatment of femoral head necrosis, and further clarify its femoral head necrotic pathway and targets, and to explore the mechanism of action related to the Wnt pathway and BMP pathways through, as a guide JPF in the clinical treatment of non-traumatic osteonecrosis of the rational use and provide an experimental basis for further drug development.Methods1. Using a 4-week-old male SD rats were extracted bone marrow adherent isolated rat primary BMSCs,1:3 ratio of passage.3-5 cells used in the experiment;2. JPF After boiling, alcohol precipitation, vacuum concentration, solvent separation system, get a different Extracts:petroleum ether, chloroform site, ethyl acetate fraction, n-butanol fraction, water part. Prepared as a crude drug containing liquid containing 1g/ml;3.3. Different concentrations (10-7～10-2mg/ml) of petroleum ether, chloroform site, ethyl acetate fraction, n-butanol fraction and water sites from JPF drugs and BMSC were incubated for 48h, MTS assay using drugs cell proliferation;4. Different concentrations (10-6～10-4mg/ml) of petroleum ether, chloroform site, ethyl acetate fraction, n-butanol fraction and water sites from JPF drug act on BMSCs for 7d or 14d, BMSCs were induced to differentiate into bone and adipogenic differentiation using osteogenic differentiation-inducing culture medium and adipogenic differentiation-inducing culture medium.detection of drugs osteogenic differentiation of BMSCs using ALP staining and ARS staining; using oil red O staining influence of drugs on BMSCs adipogenic differentiation;5. The water sites acts on BMSCs for 7d or 14d, collect cell supernatants by ELISA, cells were collected RNA using Real-time PCR to detect the role of water extraction on the expression of bone parts mark during differentiation factor ALP, BMP2, Dlx5, OCN, Runx2; ethyl acetate extract affects the expression of lipid markers during differentiation factor PPARγ, LPL and AP2;6. Join Wnt pathway inhibitor DKK-1, and BMP pathway inhibitor Noggin, and then using join pathway inhibitor DKK-1 and Noggin, by western methods to detect the expression of the downstream pathway factor, and then clear the drug’s mechanism of action.Results1. The JPF different extraction parts to culture rat BMSCs in vitro proliferationJPF effective concentration of different extracts (10-6～10-2 mg·mL-1) on cultured rat BMSCs in vitro have different levels to promote cell proliferation. In which different parts of the concentration of extracted water can significantly promote the proliferation of BMSCs (P<0.05, P<0.013或P<0.001)), and there is a concentration-dependent manner. Different concentrations of chloroform extraction sites can significantly promote the proliferation of BMSCs (P<0.05, P<0.01或P <0.001)), and there is a concentration-dependent manner. N-butyl alcohol extracted part of different concentrations on the proliferation of BMSCs also has obvious promoting effect (P<0.05, P<0.01 或 P<0.001)), only in 10-6～10-4 mg mL-1 with concentration dependence. Ethyl acetate extract parts of different concentrations on the proliferation of BMSCs have significantly promoted (P<0.05, P<0.01), no significant dose-effect relationship.2. The affection of JPF different extraction parts to cultured rat BMSCs osteogenesis differentiation in vitroJPF effective concentration of different extracts (10-6～10-4 mg/ml) on cultured rat BMSCs osteogenic differentiation as well as the different roles of the adipogenic differentiation. Among them, the water concentration-dependent parts can enhance and promote the formation of calcium nodules ALP activity (P<0.05, P<0.01 or P <0.001), and the best effects at high concentrations. At the same time there are certain adipogenic differentiation inhibition (P<0.05, P<0.01), but no significant relationship between dose. Ethyl acetate can be concentration-dependent inhibit the formation of fat cells (P<0.05, P<0.01). Chloroform site can promote the formation of calcium nodules (P<0.05, P<0.01 or P<0.001), but not obviouly for the ALP activity and the pormation of fat cells. N-butanol for osteogenic differentiation of fat are not significant.3. The affection of water extraction sites of JPF on iconic protein during cultured rat BMSCs osteogenic differentiationParts of water extractions at different concentrations (25ng/ml,50ng/ml, 100ng /ml) can significantly promote the BMSCs into expression bone alkaline phosphatase activity during the differentiation of osteogenesis, and there is a concentration-dependent manner, in which the effect of high doses of the positive drug the effect is similar. Water sites also can significantly promote the expression of β-catenin and BMP2 from BMSCs supernatant, where β-catenin is a key transcription factor of canonical Wnt signaling pathway, whereas BMP2 is a key factor of BMP signaling pathway. By studying the mRNA expression of osteogenic differentiation related factor, was found in the water portion of the middle,high dose (50ng/ml, 100ng/ml) were significantly promoted the mRNA expression of ALP, Dlx5, BMP2, Runx2 and OCN on osteoblast differentiation during early, mid, late stage (P <0.05, P<0.01), low dose (25ng/ml) has a significant role in promoting the mRNA expression of ALP and BMP2 (P＜0.05), but Dlx5, not much affect the mRNA expression of OCN and Runx2.4. JPG water extraction site to promote the mobilization and migration of rat MSCs cultured in vitro.Different concentrations of water sites (25～100 ng/ml) can effectively promote the mobilization of the migration behavior of BMSCs, low, medium and high doses can significantly promote the migration distance of BMSCs (P<0.05, P<0.001, P <0.001) and migration area (P<0.05, P＜0.05, P＜0.001).5. The mechanism of JPF water extraction site to promote the rat BMSCs osteogenic differentiation in vitro.After extracting JPF water of high concentration (100ng/ml) were added in DKK-1 and Noggin, ARS staining was found that the formation of calcium nodules were inhibited from absorbance values and stained area statistics analysis, after adding inhibitors, the formation of calcium nodules significantly reduced (P<0.05, P <0.01), suggesting the water parts play a role of osteogenesis may be through the Wnt signaling pathway and BMP signaling pathway to play a role. From the protein level experiments further confirmed this result, after the addition of inhibitior,the expression of Wnt signaling pathway downstream protein (β-catenin, Runx2) were significantly decreased, likewise, BMP signaling pathways downstream of protein (beta-catenin, Runx2, OSX) after adding inhibitor of, there is expression significantly lower.6. The affection of JPF different extraction parts to cultured rat BMSCs adipogenic differentiation in vitroJPF effective concentration of different extracts (10-6～10-4 mg/ml) on cultured rat BMSCs direction different roles to adipogenic differentiation. It was found that the water in parts of the site and ethyl acetate 10-6～10-4 mg/ml within the range, you can significantly reduce the number of oil red O staining (P<0.05 or P<0.01) cells, which ethyl there are parts of the exhibit concentration-dependent trend. Chloroform and n-butanol fraction had no significant effect. The positive control drug simvastatin can significantly inhibit the formation of fat cells (P<0.01).7. The affection of JPF ethyl acetate extraction on cultured rat BMSCs adipogenic differentiationDifferent concentrations of ethyl acetate extract (1～100ng/ml) also significantly inhibited adipogenic differentiation of BMSCs induced by adipogenic differentiation medium, and can significantly reduce the number of fat cells in the area of positive staining (P<0.05 or P<0.01), and a concentration-dependent manner. And low, medium and high doses of ethyl acetate extract can inhibit the key transcription factor of the adipogenic differentiation procession, PPARy, the initiation of transcription factor protein factor, LPL and AP2 of terminal differentiation (P<0.05, P<0.01, P 0.001) and mRNA expression (P<0.05 or P<0.01).8. The mechanism of JPF ethyl acetate extract parts to promote the rat BMSCs adipogenic differentiation in vitro.After adding Wnt pathway inhibitor, ethyl high doses of oil red O staining results showed that the number of fat cells and the area has not decreased, but there was significantly increased in the phenomenon (P＜0.05 or P<0.01). After being blocked Wnt pathway, which inhibits differentiation into the role of fat has also been blocked, suggesting that drugs may play a role through the Wnt pathway; Corresponding WB experimental results, the high-dose drug group with inhibitors of protein expression significantly increase (P<0.01 or P<0.001), and further validate the drug through its inhibition of Wnt pathway plays adipogenic differentiation.After the addition of BMP pathway inhibitors, ethyl high dose of oil red O staining showed that, the number of fat cells and a significant reduction in area appears a phenomenon (P<0.05 or P<0.01). After BMP pathway is blocked, which promote adipogenic differentiation action is also blocked, appear adipogenic differentiation was significantly inhibited in the phenomenon, the drug may be prompted by BMP pathway plays a role; Corresponding WB experimental results, the high-dose drug group with inhibitors of protein expression significantly increase (P <0.05 or P<0.01), and further validate the drug through its inhibition of BMP pathway plays adipogenic differentiation.In summary, this study by JPF of different extracts in vitro BMSCs proliferation, differentiation and migration of the study were selected to play an effective part of the role is to contribute to bone parts water, inhibiting effective part of adipogenic differentiation is ethyl acetate extract, to further explore the role of the molecular mechanisms of drugs, may be used to play efficacy by Wnt signaling pathway and BMP signaling pathways. |
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