Study On The Effect And Mechanism Of Zhonghua Dieda Pill On Promoting Fracture Healing

ObjectiveTwo animal models of zebrafish caudal fin fracture and rabbit radial fracture were used to evaluate the effect and mechanism of Zhonghua Dieda Pill on promoting fracture healing,so as to provide research basis for increasing clinical new indications and secondary development of Zhonghua Dieda Pill.Methods1.Effect and mechanism of Zhonghua Dida Pill on fracture healing in zebrafish: The fracture model was established by squeezing zebrafish caudal fin with a sharp edge knife.The fracture model was randomly divided into five groups,namely,the model group,the positive drug control group(Jiegu Qili Tablets 2 mg / tail),the low,medium and high dose groups of Zhonghua Dieda Pill(0.22,0.67 and 2 mg / tail),and the normal control group.After 7days of administration,the fracture healing effect was evaluated by alizarin red staining;Transgenic neutrophil fluorescent zebrafish was used to establish the fracture model,and the local neutrophil fluorescence intensity was collected after administration for 24 h to evaluate the anti-inflammatory effect of the drug on zebrafish fracture;Transgenic zebrafish were used to establish a fracture model.After 24 hours of administration,the vascular area of zebrafish was measured to evaluate the effect of drugs on angiogenesis in fracture site of zebrafish;The zebrafish cartilage injury model was established by dexamethasone.After 72 hours of administration,the fluorescence intensity of zebrafish craniofacial cartilage was collected to evaluate the protective effect of dexamethasone on zebrafish cartilage injury.Q-PCR was used to detect the effects of Zhonghua Dieda Pill on the expression of TNF-α,IL-1β,BMP-2 and BMP-7 genes in zebrafish with fracture,and to explore the mechanism of anti-inflammation and promoting fracture healing.2.Effect and mechanism of Zhonghua Dieda Pill on healing of radial fractures in rabbits: Seventy-two rabbits were numbered to establish a fracture model of one third of the radius.After modeling,72 rabbits were randomly divided into 4 groups: model group,positive control group,Zhonghua Dida Pill high,low dose group,with 18 rabbits in each group.The positive group was given Jiegu Qili Tablets 0.16 g / kg / d,the high dose group was given Zhonghua Dieda Pill 2.60 g / kg / d,and the low dose group was given Zhonghua Dieda Pill 0.65 g / kg / d.At the 2nd,4th and 6th week after operation,6 rabbits in each group were given blood through ear vein and their left and right radiuses were taken.The fracture healing of each group was detected by X-ray before execution.The flexural strength of fracture radius was detected by universal material testing machine.HE staining was used to observe the local repair of fracture.The expression of serum calcium and phosphorus,PINP and bALP were detected by ELISA.Micro-CT was used to observe the microscopic changes in callus during fracture healing.Fracture recovery was analyzed by three-dimensional composition.Results1.Effect and mechanism of Chinese Dieda Pill on fracture healing in zebrafish:Compared with the model control group,the promoting effect of fracture healing in the high concentration group of Zhonghua Dieda Pill was20.8 %(P < 0.05).The regression rates of inflammation in the low,medium and high concentration groups were 44.6 %,50.6 % and 52.6 %,respectively(P < 0.05),and the expressions of TNF-α and IL-1β genes were down-regulated to varying degrees(P < 0.01 & P < 0.05).The promoting effects of medium and high concentrations on angiogenesis were 22.9 % and54.0 %,respectively(P < 0.05 & P < 0.01).Zhonghua Dieda Pill played a role in cartilage repair by up-regulating the expression of BMP-2 and BMP-7genes,and the repair effect of cartilage injury in high concentration group was42.0 %(P < 0.05).2.The effect and mechanism of Zhonghua Dieda Pill on radial fracture healing in rabbits:(1)Results of X-ray examination: At the 2nd week after operation,callus formed at the broken ends of each administration group,and the bone gap decreased significantly.At the 4th week after operation,the bone gap further decreased or even disappeared.At the sixth week after surgery,the bone gap almost disappeared.(2)Biomechanical test results of callus: At the 2nd and 4th weeks after the operation,there was no significant difference between the Chinese drop pill group and the model group.At the6 th week after the operation,the flexural strength of the fracture site in the high-dose Chinese drop pill group was significantly higher than that in the model group(P < 0.05).(3)Pathological HE section detection: At the second week after operation,the callus of positive group and Zhonghua Dieda Pill group were mainly fibrous cartilage and more trabecular bone,while the trabecular bone of model group was relatively less.At the 4th week after operation,the callus in the model group began to transform into bony callus,and the callus in the positive group and the Zhonghua Dieda Pill group began to form lamellar bone.At 6 weeks after operation,the model group began to form lamellar bone.(4)Serum biochemical indexes: There was no significant difference in serum calcium and phosphorus at the 2nd and 4th weeks after operation,while PINP and bALP showed an upward trend in value,but there was no statistical difference.At the sixth week after operation,there was significant difference in serum calcium and phosphorus between the positive group and the high-dose group of Zhonghua Pill were significantly different from those in the model group.The PINP content was numerically higher than that in the model group,but there was no significant difference.The bALP content in the positive group and the high-dose group of Zhonghua Dieda Pill was significantly higher than that in the model group(P < 0.05).(5)Micro-CT result of survey: At the second week after operation,compared with the model group,the BMD and TV values of radial fracture in the high dose group of Zhonghua Dieda Pill were significantly decreased(P < 0.05).At the sixth week after operation,compared with the model group,the BMD value in the high-dose group of Zhonghua Dieda Pill was significantly increased(P < 0.05),and the TV value was significantly decreased(P <0.05).There was no significant difference in BV / TV,Tb.Sp,Tb.Th and SMI values.The results of three-dimensional microscopic composition showed that,compared with the model group,at the second week after operation,more skeletal tissues passed through the fracture ends and the thickness was thicker in the high and low dose groups of Zhonghua Dieda Pill.At the 4th week after operation,the newly generated callus pores and callus volumes in the high and low dose groups of Zhonghua Dieda Pill were significantly reduced.At the sixth week after operation,the callus volume in the high-and low-dose groups of Zhonghua Dieda Pill was almost disappeared and the remodeling of plate bone was obvious,while the callus volume in the model group became smaller and began to reshape into plate bone.Conclusion(s)1.Zhonghua Dieda Pills can promote fracture healing,anti-inflammatory,vascular and cartilage repair in fracture zebrafish.The anti-inflammatory effect may be related to down-regulating the expression of TNF-α and IL-1β,and the cartilage repair may be related to up-regulating the expression of BMP-2 and BMP-7 genes.2.Chinese Dieda Pill can promote the healing of radial fracture in rabbits,which may play a role in promoting the healing of radial fracture in rabbits by increasing serum calcium and phosphorus content and promoting the expression of PINP and bALP.