| Objective:The animal model of spinal cord injury(SCI)was established by modified ALLEN method,we investigate whether rutin can promote the recovery of neurological function in rats after SCI,improve the infiltration of inflammatory factor.Detection of rutin on effects of PI3K/AKT signal pathway after SCI,and to explore the mechanism of rutin play a neuroprotective effect after SCI.Methods:1.Animal model and grouping: In this experiment 120 female S-D rats were randomly divided into four equal groups with each group consisting of thirty rats.Sham operation group(CON group): The rats were underwent laminectomy without damage spinal cord and intraperitoneal injection of 1ml DMSO.Spinal cord injury group(SCI group): The rats according to the ALLEN method to establish the rat model of SCI(T10),and received 1mL of DMSO by intraperitoneal injection;Methylprednisolone treatment group(MP group): The pattern of spinal cord injury was the same as SCI group,the rats received intraperitoneal injection of methylprednisolone 30mg/kg;Rutin treatment group(RT100 group): The pattern of spinal cord injury was the same as SCI group,the rats received intraperitoneal injection of rutin 100mg/kg.2.Ten rats in each group were detected after spinal cord injury in 1?2,?3 weeks,the somatosensory evoked potentials(SEP)were used to detect the spinal cord function in rats,the latency and amplitude as observed index.In the 1?7?14?21?28 days after SCI,the BBB score,slope test and modified Tarlov score of behavioral ways to evaluate hindlimb motor function and body balance ability of rats.3.Animals were sacrificed 24 hours after SCI in rats.Five rats in each group were used to measure the water content of fresh spinal cord;Five rats in each group were taken fresh spinal cord for ELISA,gelatin zymography and spectral analysis.Five rats in each group were taken fresh spinal cord for Western Blot.Five rats in each group were perfused with 4% paraformaldehyde and then fixed in the same fixed solution for 24 hours.The paraffin embedded sections were used for immunohistochemical staining and HE staining.4.Immunohistochemical staining was used to determine the spinal cord of IL-1??TNF-??MCP-1?IL-10?TGF-??GFAP?IBA-1?Bax?Bcl-2 expression,HE staining was used to observe the morphological and pathological manifestations of spinal cord tissue.5.Determination of spinal cord tissue water content by using the Hsu formula,using enzyme-linked immunosorbent assay(ELISA)determination of MIP-2 in spinal cord tissue,the content of MMP-9 was measured by gelatin zymography in spinal cord tissue,using spectrum analysis method determination of IKK? kinase activity in spinal cord tissue.6.Using western blot method detected P-AKT,NF-kappa B P65,Bax,Bcl-2expression in spinal cord tissue.Results:1.The results of SEP: SCI group in the 1,2,3 week compared with CON group,the latency of SEP was prolonged,the amplitude of SEP decreased significantly;compared with SCI group,MP group and RT100 group showed latency period decline in varying degrees and amplitude was increased in varying degrees in 1,2 and 3weeks,the difference was statistically significant(p<0.05).2.BBB,slope test,the modified Tarlov function score results: In each detection point time after spinal cord injury,the rats in group SCI were significantly lower than CON group;compared with SCI group,MP group and RT100 group rats were have no obvious difference in the 1 and 7 days,but in the 14,21,28 days increased obviously,the difference was statistically significant(p<0.05).3.The results of water content in spinal cord tissue: compared with Con group,the water content was significantly higher than the control group in SCI group,the difference was statistically significant(p<0.05);compared with SCI group,spinal cord water content decreased significantly in MP group and RT100 group,the difference was statistically significant(p<0.01).4.The results of number of neurons in the spinal cord tissue: compared with the CON group,the number of neurons in the SCI group was significantly reduced,the difference was statistically significant(p<0.05).Compared with the SCI group,rutin group and MP group,reducing the numbers of neurons were inhibited,the difference was statistically significant(p<0.05).5.The results of inflammatory factors expression by Immunohistochemical:Compared with CON group,IL-1??TNF-??MCP-1were significantly higher in SCI group(p<0.05).Compared with SCI group,the expression of IL-1??TNF-??MCP-1were significantly lower in RT100 group and MP group,the difference was statistically significant(p<0.05).compared with CON group,IL-10,TGF-? levels increased significantly in SCI group,the difference was statistically significant(p<0.05);compared with SCI group,RT100 group and MP group in IL-10,TGF-?expression levels increased significantly,the difference was statistically significant(p<0.05).6.The content of MIP-2 was detected by ELISA: Compared with CON group,MIP-2 in SCI group was significantly higher,the difference was statistically significant(p<0.05),at the same time,compared with the SCI group,the MIP-2 level of MP group and RT100 group decreased,the difference was statistically significant(p<0.05,p<0.01).7.The MMP-9 activity was measured by gelatin zymography: compared with Con group,MMP-9 activity and output were significantly increased in SCI group,the difference was statistically significant(p<0.05).Compared with the SCI group,the production and activity of MMP-9 in MP group and RT100 group were significantly decreased,the difference was statistically significant(p<0.01).8.The results of GFAP and IBA-1 protein expression by Immunohistochemical:Compared with Con group,the expression level of GFAP and IBA-1 increasedsignificantly in SCI group,the difference was statistically significant(p<0.05);compared with SCI group,RT100 group and MP group could inhibit GFAP and IBA-1 expression,the difference was statistically significant(p<0.05).9.The results of IKK? activity by spectral analysis: In the CON group in the spinal cord of IKK? activity is relatively low,compared with CON group,IKK?activity increased significantly in SCI group,the difference was statistically significant(p<0.05);compared to SCI group,MP group and RT100 group significantly decreased,the difference was statistically significant(P < 0.05).10.The expression of Bax and Bcl-2 by immunohistochemical and western blot:Compared with the CON group,the levels of Bax in the SCI group were significantly increased and the level of Bcl-2 was significantly lower,the difference was statistically significant(p<0.05).Compared with SCI group,RT100 group and MP group can decrease the level of expression of Bax and increase the expression level of Bcl-2,the difference was statistically significant(p<0.05)11.Expression of p-Akt and NF-kappa B by Western blotting: compared with CON group,the expression of p-Akt and NF-kappaB P65 protein expression significantly increased in SCI group,the difference was statistically significant(p<0.05);compared with SCI group,RT100 group and MP group can reduce the expression of p-Akt and NF-K B P65 protein,the difference was statistically significant(p<0.05).Conclusions:1.Rutin can shorten the latentperiod and increase the amplitude of spinal cord injury in rats,improve the hindlimb ability and the body balance ability of rats after SCI.It has a protective effect on the neurological function of rats with SCI.2.Rutin can relieve spinal cord edema after spinal cord injury,reduced neuronal cell damage,inhibit the secretion of pro-inflammatory cytokines IL-1?,TNF-?,reduced of chemokines MCP-1,MIP-2,MMP-9,promote the expression of anti-inflammatory factor IL-10,TGF-?,inhibited inflammatory cell infiltration and activation of astrocytes and microglia,reduced inflammatory reaction.3.Rutin can reduce beta IKK? kinase activity after SCI in rats by inhibiting thePI3K/AKT signaling pathway,reduce the expression of p-Akt and NF-kappa B P65 protein after SCI,reduce inflammation;at the same time,rutin can inhibit the expression of Bax and improve the expression level of Bcl-2.Rutin may be has a protective effect on SCI rat through PI3K/AKT signaling pathway. |
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