| Objective:By establishing a rat spinal cord injury model,the potential neuroprotective effect of calcitriol in spinal cord injury was investigated,the regulatory effects of calcitriol on Nrf2 nuclear translocation and oxidative stress related factor NOX2 were determined,and whether the above indexes were involved in the motor function recovery after spinal cord injury in rats was analyzed.Methods: Ninety-six SPF adult male SD rats were randomly divided into four groups(24 rats in each group): sham operation group,spinal cord injury group(model group),2ug/kg calcitriol treatment group(treatment group),and 30 mg/kg autophagy inhibitor(3-MA)intervention group(autophagy inhibition group).The spinal cord injury model of SD rats was made using an electric spinal cord injury impactor.The sham operation group only performed laminectomy without impacting the spinal cord,and the rest of the groups were subjected to spinal cord impact treatment(Impact head size 2.5mm,Impact force 200 kdyne,Impact depth 4mm,impact compression time 1s).Rats in each group impacted by the percussion instrument were successfully modeled if they saw the flapping of both lower limbs.The relevant liquid was injected immediately after the model was successfully modeled.In each group of rats with aneurysm clamps,the fluttering of both lower limbs was deemed as successful modeling,and relevant fluids were injected immediately after the modeling.The treatment group was intraperitoneally injected with 2ug/kg calcitriol solution,the autophagy inhibition group was intraperitoneally injected with 2ug/kg calcitriol solution and 30mg/kg3-MA solution,and the model group and the sham group were injected with the same volume of normal saline,twice a day.At corresponding time points after modeling,each group was killed by cardiac air embolization.Correlative segments of spinal cord of rats in each group were dissected,and paraffin section and frozen section were made respectively,and tissue protein was extracted.BBB scores were used to observe motor function recovery after building.Hematoxylin-eosin(HE)staining was used to observe the remaining spinal cord tissue in different groups.Nissl staining was used to detect the remaining number of neurons in different groups.Immunohistochemical staining was used to detect the positive expression of Bcl2 and Bax in spinal cord tissue.Western Blot was used to detect the protein expressions of Neu N,p62,LC3 B,Keap1,Nrf2,NOX2,Bcl2 and Bax in tissues.Double immunofluorescence staining was used to detect the nuclear translocation of Nrf2 in neurons,and immunofluorescence staining was used to detect the expression of NOX2.The software Image J was used to process the HE staining images,Nissl staining images,immunohistochemical images and Western Blot images,and the relationship between their expression levels was quantitatively analyzed.Results: Behavioral scores showed that the treatment group improved motor dysfunction to some extent at 7 and 14 days after spinal cord injury(P<0.001).In terms of histology,HE staining results showed that,compared with model group,treatment group in the rat spinal cord structure restoration,neuron degeneration and white matter changes of myelin is abate,the percentage of residual spinal cord tissue was significantly higher than that of model group(P<0.001).Compared with the treatment group,the spinal cord structure in the autophagy inhibition group was significantly damaged(P<0.001).Nissl staining results showed that compared with the model group,the number of surviving neurons in the spinal cord of rats in the treatment group increased(P<0.001).Compared with the treatment group,the number of surviving neurons in the autophagy inhibition group decreased(P<0.001).Western blot analysis of neuronal markers(Neu N)showed that compared with the sham operation group,the expression of Neu N in the model group was decreased(P<0.01),the protein expression in the treatment group was increased(P<0.05),and the protein expression in the autophagy inhibition group was decreased compared with the treatment group(P<0.01).Western blot analysis of autophagy-related proteins p62 and LC3BⅡ showed that compared with the sham operation group,the expression of p62 protein in the model group was increased(P<0.05),and the protein expression of the treatment group was lower than that in the model group(P<0.01).Compared with the model group,the expression of LC3BⅡ protein in the treatment group was increased(P<0.001),and the protein expression of the autophagy inhibition group was lower than that in the treatment group(P<0.001).Western blot analysis of the upstream protein Keap1 showed that compared with the model group,the protein expression of the treatment group was decreased(P<0.001),and the protein expression of the autophagy inhibition group was increased compared with the treatment group(P<0.01).Dual immunofluorescence analysis of Nrf2 co-localization with Neu N showed that more neurons in the treatment group had nuclear displacement.Western blot was used to detect the expression of Nrf2 in the nuclear protein of the spinal cord.Compared with other groups,In the treatment group,the expression of Nrf2 protein increased significantly(P<0.001).The expression of NOX2 related to oxidative stress was analyzed by immunofluorescence,and the fluorescence intensity of NOX2 in the treatment group was decreased compared with that in the model group.Western blot analysis of apoptotic-related proteins Bcl2 and Bax showed that compared with the sham operation group,the Bcl2 protein expression in the model group was decreased(P<0.05),the protein expression in the treatment group was increased(P<0.001),and the protein expression in the autophagy inhibition group was decreased(P<0.001).Compared with the sham operation group,the protein expression of Bax in the model group was increased(P<0.001),the protein expression in the treatment group was decreased(P<0.001),and the protein expression in the autophagy inhibition group was increased(P<0.05).The results of immunohistochemical staining were consistent with those of Western blot(P<0.001).Conclusions:1.Calcitriol can attenuate the pathological damage of spinal cord tissue after spinal cord injury in rats,thereby exerting a neuroprotective effect.2.Calcitriol can promote the nuclear translocation of Nrf2 in neurons after spinal cord injury in rats by activating autophagy.3.Calcitriol can reduce the expression of oxidative stress-related factor NOX2 after spinal cord injury in rats by activating autophagy,and inhibit the oxidative stress process after spinal cord injury in rats,thereby exerting antioxidant protection. |
PDF Full Text Request