Effect Of Yishen Recipe On JAK2/STAT3 Signal Transduction Pathway In Fractured Rats

Purpose:The expression of activation factor il-2 and PDGF protein in serum was detected.To observe the effect of yishenfang on the signal transduction pathway of JAK2/ STAT3 in the process of fracture healing in rats.The expression of JAK2 and STAT3 protein was detected by immunohistochemistry.To study the mechanism of yishenfang's influence on fracture healing and to explore its effect on fracture treatment from multiple perspectives,to lay a theoretical foundation for the discovery of new therapies.Material and method:The experimental animals were SD male rats,48 in total,and purchased from liaoning changsheng biotechnology co.LTD.After the purchase,adaptive feeding in the real animal room of liaoning university of traditional Chinese medicine,and weighed after a week.The rats were randomly divided into four groups,which were normal group,model group,yishenfang treatment group,and the positive drug group.Modeling method: The open fracture model was selected and the rats were fast without water.The rats were anesthetized by intraperitoneal injection of 10% chloral(3ml/kg).The rats were lie prone on the operating table,exposing the skin of the lower limbs and buttocks of the rats on the left side and routine disinfection.In the left femur of rats,a straight incision of about 2.5cm was made.After the skin was cut,the muscle fascia of the rat was isolated and the femoral shaft was exposed.A small hole was drilled in the middle segment of the rat femoral shaft,and then the femoral shaft was broken by using hemostatic forceps and tweezers.Using 0.1 of the kirschner wire,it was inserted into the proximal medullary cavity of the shaft of the femoral shaft by the broken end of the fracture,and was worn out from the greater trochanter of the femur.Then the reposition is performed and the needle tail is inserted into the distal medullary cavity after resetting.Fixation of the hind leg,observe whether the fold is fixed well.If there is no activity,bend the end of the needle at the root of the large shaft of the femur and cut off the kirschner's needle.Rinse the area with saline,then dry with sterile gauze and suture the muscle,fascia and skin.The sham operation group was sutured at the same position in the left lower limb of the rat,and was sutured without osteotomy.After the successful model,the cage was raised,and the water was freely consumed during observation.The formal drug test was conducted a week later.Yishenfang treatment group to the yishenfang traditional Chinese medicine decoction.Each Chinese medicine is fully decocted and cooked twice,and then the drug concentration is 1g/ml.Shangkejiegupian provided by the pharmacy room of the affiliated hospital of liaoning university of traditional Chinese medicine(manufactured by dalian merlot Chinese pharmaceutical factory).The national drug,Z21021461,0.36 g,was configured with normal saline solution,and the solution was 1ml/100 g.1ml containing 0.39 mg powder.The normal group and the model group were treated with the same amount of normal saline,1 time per day(8:00-10:00AM).After 21 days of continuous gavage,rats were executed,arterial blood was taken,the serum was retained after centrifugation,and the rats were taken with limb femoral shaft and fixed after cleaning.ImagePro plus6.0 image analysis software was used to determine the average optical density value(MOD)of the positive expression cells of callus specimens.Serum IL-2 and PDGF were detected by enzyme-linked immunosorbent assay.The expression of JAK2 and STAT3 protein in bone scab was detected by immunohistochemistry,and the four groups were compared.Statistical software SPSS19.0 was used for statistical processing,and P < 0.05 was considered statistically significant.Results: 1.the open fracture of rats model building success,because of postoperative infection and gastric operation process,die,5 rats model group 2,only yishenfang treatment group 1 only,shangkejiegupian group 2 only.2.MOD determination: model group JAK2 and STAT3 positive cells number average optical density(MOD)is significantly higher than normal group(P < 0.05,P < 0.01),yishenfang treatment group,shangkejiegu piece group of JAK2 and STAT3 positive drug control group on the number of positive cells(MOD)significantly lower than the average optical density value of model group(P < 0.05,P < 0.01)was statistically significant.3.ELISA :Serum levels of IL-2 in rats in the model group were increased(P < 0.01)and serum PDGF content increased significantly(P < 0.05).Compared with model group,yishenfang treatment group,shangkejiegu piece of positive drug group had a significantly lower serum level of IL-2(P < 0.05,P < 0.01)was statistically significant,serum PDGF were significantly reduced(P < 0.05,P < 0.01)was statistically significant.4.Immunohistochemical morphological detection: The immunohistochemical results of the normal group,model group,yishenfang treatment group,and bone scab specimens of the shangkejiegu piece group were suggested: The expression of JAK2 and STAT3 protein in the model group,the yishenfang treatment group and the shangkejiegu piece group of the injured group was positive,and the expression of JAK2 and STAT3 protein in the normal group was negative.Conclusion:The experimental model was successful,and the fracture model was stable,operable and reproducible.Experimental results reveal that yishenfang's mechanism may be through inhibiting JAK/STAT pathway activation factor,interleukin 2(IL-2)and the expression of platelet-derived growth factor(PDGF),thereby inhibiting JAK2 / STAT3 signaling pathway activation.This experiment preliminary explored the yishenfang party by reduce inflammatory factor to leak,blocking fracture JAK2 / STAT3 signal transduction pathway of rats,thus to improve the fracture damage local microenvironment,yishenfang can shorten the rats with acute injury of bone healing period,speed up the process of fracture healing.