Study On The Mechanism Of Gubi Tongxiao Prescription Regulating Autophagy And Hormone Induced Femoral Head Necrosis Based On PI3K/Akt/mTOR Signal Pathway

ObjectiveThe animal model of steroid induced avascular necrosis of femoral head was established by injecting lipopolysaccharide and prednisolone into SPF mice.The effects of hormone induced autophagy on osteonecrosis and PI3 K / Akt / mTOR signaling pathway were observed,and the relationship between autophagy and the etiology of steroid induced avascular necrosis of femoral head was discussed.MethodModelingIn this study,90 20-23 g male SPF Kunming mice were randomly divided into three groups,30 in each group.Control group: simple intramuscular injection of normal saline;femoral head necrosis group: intramuscular injection of lipopolysaccharide(1mgkg)combined with prednisolone acetate(100mg / kg);gubitongxiao group:intramuscular injection of lipopolysaccharide(1mgkg)combined with prednisolone acetate(100mg / kg),and then intramuscular injection of gubitongxiao formula after injection,4 times in each group,with an interval of 1 week.The animals in each group were taken at 2,4,6 and 8 weeks after modeling.The empty bone lacuna rate was calculated under light microscope.The expression of microtubule associated protein LC3-Ⅱ was detected by immunofluorescence and Western blotting.The expression of Akt,mTOR and autophagy associated protein Beclin1 were detected by real-time quantitative QRT PCR and Western blotting.Result1.The empty bone lacuna rate of the control group was less than 12% of the normal bone,and there was no statistical significance in the four times of specimen observation.The rate of empty bone lacuna in osteonecrosis of femoral head group and gubitongxiao formula group was higher than the normal value(P < 0.05),and the value increased rapidly in the second and fourth weeks,and slowly in the sixth and eighth weeks,which was related to the rapid steroid induced osteonecrosis of femoral head.The empty bone lacuna rate,femoral head necrosis and ring death rate of gubitongxiao formula group were lower than those of steroid femoral head necrosis group.2.Cell immunofluorescence labeling Compared with the control group,the expression of microtubule associated protein LC3-Ⅱ in osteonecrosis of femoral head group and gubitongxiao formula group increased.3.Real time fluorescent quantitative RT-PCR detection The expression of Beclin1 mRNA in the control group was stable by real-time fluorescence quantitative RT-PCR,and there was no big fluctuation in the four sample examinations.The rest of the groups were based on this group.In the second week,Beclin1 mRNA increased to 16.01 times of the control group(P < 0.01),8 times of the control group in the fourth week,3 times of the control group in the sixth week,and 0.46 times in the eighth week.Compared with the control group,the expression of Akt mRNA in gubitongxiao formula group was significantly decreased;compared with the control group,the expression of mTOR mRNA in femoral head necrosis group and gubitongxiao formula group was significantly decreased.Beclin1 was activated rapidly and autophagy was induced.However,the expression of Beclin1 mRNA decreased with the progression of the disease.Beclin1 mRNA was also higher in the treatment group than that in the control group,but the expression of Beclin1 mRNA was significantly lower in the treatment group than that in the control group due to the inhibition of autophagy by gubitongxiao formula.4.Western blotting was used Compared with the control group,the expression of microtubule associated protein LC3-Ⅱ in femoral head necrosis group and gubitongxiao formula group was significantly increased(P < 0.01),and the expression of p-Akt group and p-mTOR protein was significantly decreased(P < 0.01).Discuss1.Avascular necrosis of the femoral head is common in young people,it is very easy to make people disabled,more than 70% of people will be secondary to osteoarthritis.The use of hormone is a major factor in avascular necrosis of the femoral head.The reason why there are endless clinical treatment programs for femoral head necrosis,and there is not a good treatment program to prevent the occurrence and development of the disease,is because there is no clear explanation of the cause of the disease.This paper is to clarify the relationship between autophagy and osteonecrosis,and the role of autophagy in the development of steroid induced osteonecrosis of the femoral head.Jia et al. have shown that the pathogenesis of steroid induced avascular necrosis of the femoral head is closely related to osteocyte apoptosis and autophagy.2.In this experiment,we found that the expression of microtubule associated protein LC3-Ⅱ,Beclin1 mRNA and Beclin1 protein increased rapidly in the femoral head necrosis group after hormone injection,and increased rapidly in the second and fourth weeks,and decreased in the sixth and eighth weeks,indicating that hormone can induce autophagy quickly.The expression of Akt mRNA and mTOR mRNA decreased,and hormone could inhibit the reverse regulation of PI3 K / Akt / mTOR signaling pathway and induce autophagy.After intervention,gubitongxiao decoction could significantly affect the expression of microtubule associated protein LC3-Ⅱ,autophagy associated protein Beclin1,Akt mRNA and mTOR mRNA,and inhibit autophagy.