| Objective to establish an animal model of alcohol-induced osteonecrosis of femoral head,and to explore whether necroptosis and apoptosis of bone cells are involved in the pathological process of femoral head necrosis through the intervention of necroptosis inhibitor(Nec-1s)and apoptosis inhibitor(z VAD).Methods(1)model establishment: after accepted strong distilled spirits(56% ethanol)10 m L/(kg/d)gavage for 4,8 and 12 weeks,the specimens of unilateral femoral head were taken for histopathological observation,conduct the next experiment after determining the success of modeling.(2)Drug intervention: 60 healthy four month old male SD rats were divided into four groups: blank control group(12),model group(16),Nec-1s group(16)and z VAD group(16).The blank control group was not given alcohol gavage.The model group,Nec-1s group and z VAD group were given alcohol gavage according to the above methods.Nec-1s group was injected intravenously with Nec-1s,z VAD group was injected intravenously with z VAD,and the model group and blank control group were injected intravenously with the same amount of normal saline.The body weight was weighed and micro CT scanning was performed at four time points of continuous intervention 4,8 and 12 weeks,and bilateral femoral head specimens were taken for following detection.The bone mineral density(BMD),bone volume/ total volume(BV / TV),trabecular thickness(TB.Th)and trabecular separation(TB.SP)of the right femur were measured.After12 weeks intervention,He staining was used to analyze the morphological changes of bone tissue,and the number of osteocyte(n.ot)were calculated.TUNEL method was used to detect the apoptosis of bone cells,and immunohistochemistry and Western blot were used to detect the expression of Rip1,RIP3 and Caspase 8 in bone tissue.Spss20 0 for used for statistical analysis.Results the modeling results showed that the rats in the model group had anorexia,dull hair,decreased activity,arched back shape,slow response,increased liver volume and yellowish brown,thin cartilage layer with obvious necrotic lesions under the microscope,obvious "peripheral" shape of bone cells,sparse bone trabeculae,disordered arrangement and increased structural spacing,The incidence of vacant bone lacunae and osteonecrosis was significantly higher than that of the control group,indicating that this method was successful.Intervention results(1)Body weight: there was no significant difference in body weight between Nec-1s group,z VAD group and blank control group(P > 0.05),and it was significantly higher than that in model group(P < 0.05).(2)Micro CT scanning results: there was no significant difference in BMD,bone volume/ total volume(BV /TV),trabecular thickness(TB.Th),number of rat osteocytes(N.ot)between Nec-1s group,z VAD group and blank control group(P > 0.05),and it was significantly higher than that in model group(P < 0.05);There was no significant difference in trabecular resolution between Nec-1s group,z VAD group and blank control group(P > 0.05),and it was significantly lower than that in model group(P < 0.05).(3)He staining: in the model group,bone trabeculae sparse and empty bone lacunae increased at 4 weeks;At 8 weeks,the bone trabeculae became thinner and thinner,the structure was disordered,a few bone trabeculae were broken,and necrotic lesions were seen;At 12 weeks,the bone trabeculae disappeared,accompanied by typical necrotic lesions.The nucleus was deformed into a crescent shape,showing "peripheral" changes and filled with connective tissue.There was no significant change in HE staining results in Nec-1s group,z VAD group and blank control group.It can be seen that the bone trabeculae were basically arranged orderly,the bone structure was basically regular,the morphology of bone cells was normal,the bone marrow tissue in the medullary cavity was normal,there was no obvious thinning or thinning of bone trabeculae,and there were no empty bone lacunae.(4)TUNEL test results: there was no significant difference in the percentage of osteocyte apoptosis between Nec-1s group,z VAD group and blank control group(P > 0.05),and it was significantly lower than that in model group(P < 0.05).(5)Results of immunohistochemistry and Western blot: there was no significant change in the expression of RIP1,RIP3 and caspase-8 in the blank control group(P > 0.05);Theexpression of RIP1,RIP3 and caspase-8 in the model group increased significantly(P < 0.05).The expression of RIP1 in Nec-1s group had no significant change,and there was no significant difference compared with the blank control group(P > 0.05).The expression of RIP3 and caspase-8 in Nec-1s group was significantly higher than that in the blank control group(P > 0.05).There was no significant change in the expression of caspase-8 in z VAD group and there was no significant difference compared with the blank control group(P > 0.05).The expressions of RIP1 and RIP3 in z VAD group were significantly higher than those in the control group(P < 0.05).Conclusion(1)the rats with alcohol-induced osteonecrosis of femoral head can be successfully prepared by giving rats a strong distilled spirits(56% ethanol)10 m L/(kg/ d)for 12 weeks.(2)necroptosis and apoptosis are both involved in the death of osteoblast;Osteoblast inhibitor(Nec-1s)can inhibit the osteoblast necroptosis by inhibiting the expression of RIP1,but has no effect on RIP3;Apoptosis inhibitor(z VAD)can inhibit the osteoblast apoptosis by inhibiting the expression of caspase-8,but has no effect on RIP1 and RIP3;(3)Necroptosis is one of the main pathways of osteocleptosis when the caspase dependent apoptotic pathway is inhibited. |
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