Gushi Capsule Prevents Steroid-induced Osteonecrosis Of The Femoral Head By Stabilizing Apoptosis Through Mitochondrial Pathway

Research objectiveSONFH(Steroid-induced osteonecrosis of the femoral head)is a very common and intractable disease in clinical practice,mostly caused by the massive use of glucocorticoids in clinical practice.Gushi Capsule is an efficacious traditional Chinese medicine for treating steroid-induced osteonecrosis of the femoral head.It has been used in clinical practice for an extended period;however,the underlying mechanism of therapeutic action for steroid-induced osteonecrosis of the femoral head remains unclear.In this study,we established a New Zealand white rabbit model and established a traditional Chinese and western medicine intervention group for intervention,with three main purposes:(1)successfully established a New Zealand white rabbit SONFH model using MPS(methylprednisolone)and LPS(lipopolysaccharide);(2)investigating the microscopic mechanisms of Steroid-Induced Femoral Head Necrosis(3)verified the mechanism of Gushi Capsule in regulating mitochondrial pathway apoptosis in the treatment of SONFH.Research methodsWe used New Zealand white rabbits to construct an in vivo model of SONFH and adaptively fed them under standard conditions for one week.According to experimental requirements,the experimental rabbits were divided into a control group,a model group,and low,medium,and high concentrations of traditional Chinese medicine groups and a Western medicine group.Except blank group,the other groups of rabbits were injected with LPS 10 μg/kg at 1-2 days to simulate the inflammatory state.MPS 25 mg/kg was injected into the right gluteus maximus muscle at 2-4 days to induce osteonecrosis of the femoral head.Different concentrations of Chinese and western medicine were intervened from the 5th day.The drug suspension was intragastrically administered after 2 hours of drug fasting every day.The control group was intragastrically administered with normal saline for4 weeks.During the intervention,the hip joints of rabbits in each group were scanned by MRI at 2,4,and 8 weeks to observe the morphological changes of the femoral head,and then the experimental animals were sacrificed to collect bilateral femoral head specimens.After fixation,decalcification,dehydration,embedding and sectioning of the left femoral head specimens,some specimens were stained with hematoxylin and eosin to observe the morphological changes in the trabecular bone as well as the number of empty lacunae,and TUNEL staining was used to examine the necrosis of osteocytes.The right femoral head specimens were powdered after freezing in liquid nitrogen,and some of them were extracted for total protein and total RNA using RIPA and Trizol,and then their apoptotic gene expression was detected by QRT-PCR and Western blotting.Research result1.LPS and MPS can successfully establish an SONFH model in New Zealand White rabbits,with shorter modeling time and higher success rates compared to traditional methods.2.Cell morphology observation showed that Gushi Capsule could inhibit the formation of empty lacunae and stabilize the morphology of trabecular bone.3.TUNEL staining showed that Gushi Capsule could inhibit osteocyte apoptosis induced by MPS.4.The results of protein imprinting showed that Gushi Capsule could inhibit osteocyte apoptosis by inhibiting the release of apoptosis-related proteins in mitochondrial pathway,and its effect was linearly related to the dosage and administration time of Gushi Capsule.5.The results of QRT-PCR showed that the application of Gushi Capsule inhibited the expression of apoptotic genes BAX,Caspase-8 and Cytc,and enhanced the expression of anti-apoptotic gene BCL-2.Conclusion1.LPS and MPS can successfully establish SONFH model in New Zealand white rabbits.2.MPS induces osteocyte apoptosis through the mitochondrial pathway,which disrupts bone microcirculation and further leads to osteonecrosis of the femoral head.3.Gushi Capsule can prevent the occurrence of SONFH by inhibiting osteocyte apoptosis induced by MPS,which is of great significance for various diseases treated with hormone therapy in clinical practice.