| Objective:Along with the aging society developing, Osteoporosis(OP) becomes one of health issues which attract extensive attention worldwide. The rational treatment of OP should include both drug and exercise. Because of the mechanism of mechanotransduction in osteoblasts is still unclear. It is difficulty for the clinicians to guide patients exercise with the correct way. Meanwhile, conventional drug therapies have both pros and cons. For example, long-term use of estrogen therapy increases the possibility of breast and endometrial cancers. It is very important to find and develop new agents which have high recovery efficiency of bone mass without side effects. In current study, we explored the role of the two members of MAPK family, ERK5 in fluid shear stress(FSS) induced osteoblast differentiation and p38 MAPK in Radix Hedysari polysaccharides (HPS) stimulating osteoblast differentiation which would not only be helpful for development of bone tissue engineering but also provide new drug discovery targets for the treatment of OP.Methods:Part â… :MC3T3-E1 osteoblastic cells were subjected to intermittent FSS and steady FSS respectively. The MEK5/ERK5 pathway was selectively inhibited by BIX02189. Then investigating which shear pattern was more effective for promoting osteoblast differentiation and The MEK5/ERK5 pathway mediates osteoblast differentiation promoted by intermittent FSS;Partâ…¡:The crude polysaccharide HPS was isolated from the dried roots of Hedysarum polybotrys by hot-water extract in 45% yield. Then the concentrated supernatant was precipitated with 95% ethanol to obtain three crude polysaccharides accordingly. The purified HPS3d was obtained by anion-exchange column from Radix Hedysari and identified the structural characteristics of HPS3d. The osteoblasts was treated with different concentrations of HPS3d at 72 h of culture and the p38 MAPK was selectively inhibited by SB203580, p38 MAPK regulated HPS3d stimulated osteoblast differentiation was investigated by MTT, RT-PCR and Western blot.Results:Partâ… :(1) Both steady FSS and intermittent FSS could up-regulate ERK5 phosphorylation, ALP activity and the expression of OPN and OCN compared with the control. Furthermore, the levels of ERK5 phosphorylation, ALP activity and the expression of OPN and OCN were higher in cells treated with intermittent FSS than those in cells treated with steady FSS.(2) Phosphorylation of ERK5 was markedly up-regulated after intermittent FSS in contrast to the unloaded control and was nearly completely blocked after incubation with 10 mM BIX02189 for 2 h. Intermittent FSS up-regulated ALP activity and the expression of OPN, OCN and Runx-2 compared with the control. When osteoblasts were incubated with BIX02189 before exposure to intermittent FSS, these effects were all reduced. However, the ALP activity and expression of OPN, OCN and Runx-2 in unloaded cells were not affected by BIX02189 (p>0.05).Part II:(1) In the present study, the purified HPS3d was obtained by anion-exchange column. It consisted of 94.38% polysaccharide,3.40% protein and 13.30% uronic acid. The molecular weight was measured to be 84.6 kDa. The backbone consisted of galactopyranose and galacturonopyranose, and the side chains were composed of glucopyranose, rhamnopyranose and arabinofuranose. The result of elemental analysis of HPS3d revealed that there were mainly C (38.18%, in mass), H (5.66%), O (53.91%), S (1.73%). The backbone structure of HPS3d mainly contained galactose and galacturonic acid and branch chains mainly contained arabinose.(2) The osteoblasts was treated with different concentrations of HPS3d (control,0.5 to 32 mg/ml) at 72 h of culture. The results showed the viability of osteoblasts was not affected by HPS3d (0.5 to 8.0 mg/ml). Interestingly, higher concentrations(16 and 32 mg/ml) resulted in a decrease in cell viability.(3) The osteoblasts was treated with different concentrations of HPS3d (control,0.5 to 8 mg/ml) at 72 h of culture. The ALP activity and the mRNA expression of OPN, OCN and BSP were not affected when the concentration was 0.5 mg/ml. HPS3d (1.0 to 4.0 mg/ml) concentration-dependently enhanced the ALP activity and the mRNA expression of OPN, OCN and BSP and not continuously enhance when the concentration was8.0 mg/ml (P> 0.05).(4) A significant increase in Runx-2 and Osterix mRNA expression were observed starting at 1.0 mg/ml and reached 5.2 and 6.6-times increase, respectively, at 4.0 mg/ml when compared with control after 72h incubation by HPS3d. The Western blot analysis also showed that the protein expression of Runx-2 and Osterix were significantly unregulated by HPS3d at 2.0 and 4.0 mg/ml.(5) The osteoblasts was treated with different concentrations of HPS3d (control,0.5 to 8 mg/ml) at 72h of culture. The mRNA expression of BMP2 were concentration-dependently enhanced by HPS3d(1.0 to 4.0 mg/ml). The Western blot showed that the phosphorylation level of SMAD1/5/8 were significantly unregulated by HPS3d at 2.0 and 4.0 mg/ml compared with control.(6) The osteoblasts was treated with HPS3d at 72h of culture. Compared with control group, HPS3d could up-regulate the p38 MAPK phosphorylation level and was down-regulated with p38 MAPK selective inhibitor SB203580. Further study showed that the HPS3d induced ALP activity and the mRNA expression of OPN, OCN and BSP were also down-regulated while SB203580 presented.Conclusion:(1) Both 12dyn/cm2 steady FSS and intermittent FSS could stimulate osteoblasts differentiation compared with the control. Furthermore, intermittent FSS is a more intense stimulus than steady FSS for osteoblast differentiation.(2) Besides ERK1/2 and p38, the MEK5/ERK5 pathway also mediates FSS promotes osteoblast differentiation. The MEK5/ERK5 pathway regulates FSS-induced Runx-2 expression, which initiates the transcription of osteogenic genes to promote osteoblast differentiation.(3) The purified HPS3d was obtained by anion-exchangecolumn. HPS3d consisted of 94.38% polysaccharide,3.40% proteinand 13.30% uronic acid. The molecular weight of HPS3d was 84.6 kDa. The backbone of HPS3d mainly consistedof galactopyranose and galacturonopyranose, and that the sidechains were composed of glucopyranose, rhamnopyranose andarabinofuranose.(4) HPS3d up-regulated ALP activity and the expressionof other osteogenic marker genes and stimulated osteoblast differentiation. In addition, HPS3d increased the expression and phosphorylation level of Runx-2, Osterix, BMP-2 and SMAD1/5/8 respectively. These findings suggest that HPS3d stimulates osteoblast differentiation by activation of BMP-2/SMAD/Runx-2/Osteri pathway.(5) HPS3d could activate p38 MAPK. And when p38 MAPK was selectively inhibited, the HPS3d induced osteoblast differentiation was suppressed. Indicated that p38 MAPK regulated the osteoblast differentiation which was induced by HPS3d. |
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