Effects Of Bone Restorative Pill On The Expression Levels Of Serum-related Cytokines And Femoral Head Tissue Cbfα1 And Osterix In Rats With Steroid-induced Osteonecrosis Of The Femoral Head

Objective: To explore the effect of bone restorative pill on the expression levels of serum related cytokines and femoral head tissue Cbfα1 and Osterix in steroid-induced femoral head necrosis rats.Method:This experiment in male SD rats as the research object,randomly selected 15 rats as blank control group(NC group),the experimental group 75 lipopolysaccharide combined GC muscle injection is adopted to establish the early SONFH.From a random sample were executed by air embolism method in 8 rats and remove the bilateral femoral head specimens made pathological section,confirmed the early SONFH building success,and then to group.The remaining SONFH rats model according to random number table method is divided into 5 groups: 11 rats in hormone Model group,raw bone restorative pill high dose group,dose groups in bone restorative pill,raw bone restorative pill low dose group 10 each,Positive control group 11.According to the advance of experimental design for drug lavage treatment,lavage closely observe and record each SD rats during the change of the general situation,until the end of therapy on the dosing 8 weekend after 2 h,groups of rats weighing,take the blood,after centrifugal separation of serum and preserved in-20℃.Put to death by air embolism method in rats,aseptic conditions with surgical scissors and a scalpel to take out the bilateral femoral head specimens,remove cartilage,physiological saline rinse,dry filter paper,weighing on the left side of the femoral head specimens after the completion of rapid in liquid nitrogen frozen after the night shift to-80℃refrigerator,the m RNA and protein expression levels of Cbfα1 and Osterix factors were determined by PCR and Western blot,the right side of the femoral head specimens fixed in4% paraformaldehyde solution for histopathological detection after 24 h.Using ELISA method to detect the rat serum inflammatory cytokines(IL-1β,IL-6,TNF-α),serum alkaline phosphatase(ALP),serum calcium(S-Ca),serum phosphorus(S-P),osteocalcin(BGP),calcitonin(CT),tartrate resistant acid phosphatase(TRACP)and so on the content of bone metabolism related indicators.Results:(1)In the rat behavior observation: The raw bone restorative pill lavage treatment to 8th weekend,Group SGZZ-H,group SGZZ-M,group SGZZ-L and group JGSW compared with Model group mental condition,activity,diet drink,hind leg muscle and limp condition are improved to varying degrees,Among them,the symptoms of SGZZ-H group and JGSW group improved more obviously,but the NC group did not back to normal.(2)Specimens of femoral head morphological observation: Model group chapping,defect is apparent in the femoral head and poor blood supply,has the show of osteoporosis and necrosis,Group SGZZ-H,group SGZZ-M,group SGZZ-L and group JGSW compared with Model group are improved to varying degrees.(3)The femoral head histopathologica-l observation: Model group of bone cells empty pit rate compared with NC group difference was statistically significant(P<0.001),the drug lavage treatment to 8weekend after completion of materials,Group SGZZ-H,group SGZZ-M and group JGSW bone cells empty pit rate compared with Model groups all have different degree to reduce,in group SGZZ-H and group JGSW bone cells empty bone pit rate reducing the most significant.Group SGZZ-H and group JGSW empty bone pit rate there was no statistically significant difference compared(P>0.05).(4)The m RNA expression levels of Cbfα1 and Osterix were detected by PCR:Compared with NC group,the m RNA expression levels of Cbfα1 and Osterix in bone tissue of Model group decreased significantly(P<0.001);Compared with the Model group,the treatment group rats bone Cbfα1,Osterix m RNA expression level increased to some extent(P<0.05),the m RNA expression levels of Cbfα1and Osterix in bone tissue of rats in SGZZ-H group,SGZZ-M group and JGSW group increased significantly(P<0.001),the m RNA expression level of Osterix in bone tissue of SGZZ-H group and JGSW group had no significant difference compared with NC group(P>0.05),and the m RNA expression levels of Cbfα1 and Osterix in bone tissue of SGZZ-H group and JGSW group were not significantly different(P>0.05).(5)Western blot was used to detect the protein expression levels of Cbfα1 and Osterix: Compared with NC group,the expression levels of Cbfα1 and Osterix in bone tissue of rats in Model group decreased significantly(P<0.001);Compared with the Model group,the treatment group rats bone tissue of Cbfα1 and Osterix level of protein expression was also increased to different extent(P<0.05),among them,the protein expression level of Cbfα1 in bone tissue of SGZZ-H group,SGZZ-M group and JGSW group increased most obviously(P<0.001),the expression level of Osterix protein in bone tissue o f rats in SGZZ-H group,SGZZ-M group,SGZZ-L group and JGSW group increased most obviously(P<0.001),compared with NC group,the protein expression levels of Cbfα1 and Osterix in bone tissue of SGZZ-H group and JGSW group had no significant difference(P>0.05).there was no significant difference in protein expression levels of Cbfα1 and Osterix between SGZZ-H group and JGSW group(P>0.05).(6)Serum levels of IL-1β,IL-6,TNF-α,ALP,S-Ca,S-P,BGP,CT and TRACP were detected by ELISA: The expression levels of serum IL-1β,IL-6,TNF-α,ALP,CT and TRACP in Model group were significantly higher than those in NC group(P<0.001),compared with NC group,the expression levels of S-Ca,S-P and BGP in serum of Model group decreased significantly(P<0.001),compared with the Model group,the expression levels of serum IL-1β,IL-6,TNF-α,S-Ca,S-P,BGP,ALP,CT and TRACP in each treatment group were improved to some extent,the expression levels of serum IL-1β,IL-6,TNF-α,ALP,CT and TRACP in SGZZ-H group,SGZZ-M group and JGSW group decreased most obviously(P<0.01),the expression levels of serum S-Ca,S-P and BGP in SGZZ-H group and JGSW group increased most obviously(P<0.001).There was no significant difference in serum IL-1β,IL-6,TNF-α,S-Ca,S-P,BGP,ALP,CT and TRACP expression between SGZZ-H group and JGSW group(P>0.05).Conclusion:(1)Bone restorative pill can inhibit the expression of IL-1β,IL-6,TNF-α,ALP,CT and TRACP,and promote the expression of S-Ca,S-P and BGP to improve the bone metabolism,the prevention and treatment of early SONFH.(2)Osteoblast specific transcription factor Cbfαl/Osterix expression level is closely related to the early SONFH lesions in the process,raw bone restorative pill by raising bone Cbfα1/Osterix expression level to improve the activity of osteoblasts.