The Effect Of Wanshangheji Compound In The Healing Of Mouse Tibia Injury Model

Objective:investigate the enhancement of mouse tibia injury healing in vitro and in vivo by Wanshangheji compound.Material and Method:in vitro, Macrophages from L-1β-luciferase transgenic mice were challenged with Wangshangheji compound (WSHJ). Mouse primary osteoblastic cells were incubated with different type of medicated rat serum prepared with WSHJ. In vivo:A mouse tibia injury model was established. Micro CT was used for follow-up of healing in treatment-naive tibia injury mice. Serum alkaline phosphatase, ionized calcium (Ca2+) and inorganic phosphorus (IP) were measured at day20after different drugs administered. The essential amino acids in mouse serum at day10and day20were quantitated by HPLC. The alkaline phosphatase (Alp), ionized calcium, inorganic phosphorus and essential amino acids in WSHJ were measured as control. The expression of VEGF in the tibia tissue was detected by immunohistochemistry and western blot.Result:WSHJ stimulated the expression of luciferase significantly (p<0.05) higher than LPS in the macrophages from L-1β-luciferase transgenic mice. There was no proliferation effect in medicated rat serum treated mouse primary osteoblasts. But the medium alkaline phosphatase level in10%rat medicated serum was significantly (p<0.05) higher than the control group. in vivo:Micro CT analysis showed an increasing trend in BMC,BMD and Calib.Tb.Th. after tibia injury while Calib.Tb.Sp. decreasing. Tb.N was low after tibia injury. But there was no any trend in different time points. BV/TV was increasing after the injury. BS/BV increased at first and decreased subsequently. All the administered groups showed a lower alkaline phosphatase level than the control group (p<0.05, p<0.01, p<0.001, respectively). Ca2+in the group of WSHJ and OGP groups were stable and significantly lower than the control. But the group of combination of WSHJ&OGP showed a higher level of Ca2+than the control. All the administered groups showed a lower IP level than the control. Serum essential amino acids test showed arginine, leucine, leucine and proline were significantly higher than in the control group at day20. But there were no apparent changes at day10. IH and WB showed an elevated VEGF expression of WSHJ administered group as compared to control. Conclusion:WSHJ up-regulated the expression of IL-1β to improves bone formation and mineral accretion. There was no evidence of proliferative effect in mouse primary osteoblasts incubated with rat WSHJ medicated serum. But Alp increased in the medium of osteoblasts incubated with10%WSHJ medicated (q.h×4) serum. Micro CT showed dynamic changes during the healing of tibia healing in vivo. WSHJ showed some inhibitory effect on the bone dissolving process. It promoted osteogenesis and accelerated healing progress. WSHJ increased the level of bone related amino acids. The accelerated healing effect of WSHJ was involved with the up-regulated expression of VEGF and increasing angiogenic activity at the bone injury site. This pilot study provided some direct and objective evidence of acceleration fracture healing effect of WSHJ.