Protective Effects And Its Mechanism Of Tanshinone IIA On Acute Spinal Cord Injury In Rats

Objective:To explore the possibility whether Tanshinone IIA(TIIA) could completely or partly replace methylprednisolone(MP) or reduce the amount of MP in the management of ASCI in order to obtain a protective effect on nerve as similar as MP while reducing its side effect through studying the protective effect on spinal cord by TIIA or therapeutic alliance of TIIA with MP in the treatment. At the same time, it was taken that researches of TIIA on Rho A/ROCKII/MLC signaling pathways after ASCI to show the mechanism of TIIA on ASCI in rats. MethodsPart One: Establishment of rats’ cerebral astrocytes and spinal cord neurons co-culture system in two layers.The co-culture system was established by SD rats’ cerebral astrocytes and spinal cord neurons, and the cells was identify the method of immunofluorescence staining for further research.Part Two: The protective effects of combine TIIA with MP after ASCI in ratsIn vitro, spinal cord neurons damage model was established. MTT assay was used to determined the optimal concentrations of TIIA on neurons and 10μM TIIA neuron culture solution was chosed for the optimal concentrations. Cells was divided into 5 groups, Sham groups: blank control group; ASCI group: damage control group, neurons was damaged by scratch; TIIA groups: neurons was damaged by scratch, and 10μM TIIA neuron culture solution was used; MP groups: neurons was damaged by scratch, and 1μM MP neuron cell culture solution was used; TIIA + MP groups: neurons was damaged by scratch, and 10μM TIIA +0.5μM MP neuron culture solution was used. Cells were cultured for 24 hours and were collected for analysis. Western Blot analysis was used to detect the expression of Caspase-3, Bcl-2, and Bax after ASCI.In vivo, Allen weight drop method was used to creat ASCI model in rats. According to the experimental design, 60 SD rats were divided into 5 groups, Sham groups: blank control group, T9 lamina rexcision was taken, and normal breeding; ASCI group: damage control group. T9 lamina resection and ASCI model was taken, normal breeding; TIIA groups: T9 lamina resection and ASCI model was taken, rats were injected with TIIA 30mg/kg/d drug treatment; MP groups: T9 lamina resection and ASCI model was taken, rats were injected with MP 30 mg/kg drug treatment; TIIA + MP group: T9 lamina resection and ASCI model was taken, rats were injected with TIIA 30 mg/kg/d and MP 20 mg/kg drug treatment. BBB score was used to evaluate recovery of motor function of rats. RT-PCR analysis was used to detect Caspase-3 m RNA and NF-κB m RNA expression at day 1.At the sametime, Western Blot analysis was used to detect NF-κB expression in spinal cord tissues at day 1 and Caspase-3, Bcl-2 and Bax expression in spinal cord tissue. SOD’s activity and MDA content was detecte at day 1 and day 7 in the rats’ spinal cord tissue. Double immunofluorescence stain analysis was used to test the expression of Caspase-3 in spinal cord neurons in rats at day 3.Part Three: The influence of TIIA on Rho A/ROCKII/MLC signaling pathways after ASCI in ratsIn vitro, spinal cord neurons damage modelwas established, and the cells were divided into 5 groups, Sham groups: blank control group; ASCI group: damage control group, neurons were damaged by scratch; TIIA groups: neurons were damaged by scratch, and 10μM TIIA neuron culture solution was used; Fasudil groups: neurons were damaged by scratch, and 10μM Fasudil culture solution were used; Cell were collected for analysis after 24 hours. RT-PCR analysis was used to detect Rho A m RNA and ROCKII m RNA expression at day 1 and Western Blot analysis was used to detect the expression of Rho A、ROCKII、MLC and p-MLC 24 h after ASCI in neurons.In vivo, Allen weight drop method was used to creat ASCI model in rats. According to the experimental design, 32 SD rats were divided into 4 groups, Sham groups: blank control group, T9 lamina rexcision was taken, and normal breeding; ASCI group: damage control group. T9 lamina resection and ASCI model was taken, normal breeding; TIIA groups: T9 lamina resection and ASCI model was taken, and rats were injected with TIIA 30mg/kg/d drug treatment; Fasudi groups: T9 lamina resection and ASCI model was taken, and rats were injected with Fasudi 10mg/kg drug treatment; BBB score was used to evaluate recovery of motor function of rats. RT-PCR analyse was used to detect spinal cord tissues Rho A m RNA and ROCKII m RNA expression at day 1 and Western Blot analyse was used to detect Rho A、ROCKII、MLCand p-MLC expression at day 1 and day 7 in spinal cord issues. ResultsPart One: 1.1 In the microscope,the shape of cell body of primary astrocytes culture4 h after inoculation is circular, pervious to light quality, and there was no obvious bulge out. After 24 h,astrocytes sticked to wall, located in the underlying culture bottle, andcell body became flat, triangular, lozenge or polygonal, part of the cell became multiple stubby protuberant.The cells fused with each other about 70%- 80% after 7-9 days and they could be represented.1.2 The represented astrocytes sticked to wall quickly in 1 to 2 hours after inoculation. At the second day, the cell body becamebig and irregular shape. After 4-5 days, cells contacted each other.1.3 The primary culture of spinal cord neurons was observed sticked to wall under microscope after 4 h after inoculation, and cellswere circular, pervious to light quality, and there were no obvious bulge.At the second day, most of neurons stick to wall.Part Two: 2.1 After ASCI, rats’ BBB score was 0 point. At day 1 and day 2, rats’ BBB score was significantly higher in MP group than TIIA group(P<0.05), and there was no significant difference between MP group and TIIA + MP group(P>0.05). From day 5 to day 7, rats’ BBB score in TIIA group and TIIA + MP group were higher than MP group(P<0.05).2.2 At day 1 after ASCI, Caspase-3 m RNA and NF-κB m RNA expression level significantly higher in ASCI group than other groups(P<0.05). and there was no significant difference between MP group and TIIA + MP group(P>0.05).2.3 At day 7 after ASCI, Bax and Caspase-3 expression level decreased significantly in MP group and TIIA + MP group than in ASCI group(P<0.05), and Bcl-2 expression level was significantly increased(P<0.05). Bcl- 2 expression in TIIA group had no significant difference with ASCI group(P>0.05), while Bax and Caspase-3 expression level in TIIA group decreased obviously compared with ASCI group(P<0.05). Bax and Caspase-3 expression level in TIIA group and MP group increased significantly compared with TIIA + MP group(P<0.05), while the Bcl-2 expression level decreased significantly(P<0.05).Within the spinal cord neurons, the Bax and Caspase-3 expression level in ASCI group increased significantly compared with the rest ohters groups(P<0.05),while the Bcl-2 expression level decreased significantly(P<0.05). The Caspase-3, Bax and Bcl-2 expression level in MP group had no significant difference with the TIIA + MP group(P>0.05). Bcl-2 expression level in TIIA group decreased significantly compared with TIIA + MP group(P<0.05), while Caspase-3 and Bax expression level was no significant difference(P>0.05). Compared with ASCI group, the ratio of Bcl-2/Bax increased significantly in rats’ spinal cord tissues and neurons in all drug therapy group(P<0.05), and Bcl-2/Bax ratio in rats spinal cord tissues and neurons in TIIA group decreased significantly compared with MP and TIIA + MP group(P<0.05). The Bcl-2/ Bax ratio in TIIA + MP groups had no significant difference with MP group(P>0.05).2.4 At day 1 and day 7 after ASCI, SOD content increased significantly in MP group compared with ASCI group(P<0.05), while the MDA content decreased significantly compared with ASCI group(P<0.05). The content of MDA in TIIA + MP group significantly reduced compared with the TIIA group(P<0.05), while the content of SOD had no significant difference with the TIIA group(P>0.05). SOD content and content of MDA in MP group had no significant difference with TIIA + MP group(P>0.05).2.5 At day 1 after ASCI, NF-κB expression levels were significantly higher in ASCI group than other groups(P<0.05). NF-κB espression in TIIA+MP group was lower than in MP group, but there was no significant difference(P>0.05).2.6 According to the result of MTT assay, 10 μM TIIA was choiced asthe best concentration of TIIA to cultivate neurons in follow-up experiments. The OD value in neurons in ASCI group 24 hours after ASCI dropped significantly compared with other groups(P<0.05).The OD value of neurons is higher in TIIA + MP group than MP group, but no significant difference(P>0.05).2.7 At day 3 after ASCI, immunohistochemical experiment showed that the amount of the expression of Caspase-3 in the rats in TIIA group has no significant difference with in ASCI group(P>0.05), while the amount of the expression of Caspase-3 decreased significantly in MP group and TIIA + MP spines compared with ASCI group(P<0.05). There was no significant difference between MP group and TIIA + MP group(P>0.05).Part Three: 3.1 There was no significant difference of BBB score between TIIA group and Fasudil group all time(P>0.05). From day 2 to day 7, BBB scores in TIIA group and Fasudil group rats were significantly higher than the ASCI group(P<0.05).3.2 The expression of Rho A and ROCKII level in neurons in ASCI group was significantly increased compared with the other groups(P<0.05). ROCKII expression in neurons in Fasudil group had no significant difference with in TIIA group(P>0.05), while the expression of Rho A in TIIA group significantly reduced compared with Fasudil group(P<0.05).At day 1 and day 7 after ASCI, Rho A and ROCKII expression in rats in ASCI group were significantly higher than other groups(P<0.05). At day 1 after ASCI, Rho A and ROCKII express in rats in Fasudil group were ignificantly increased compared with TIIA group(P<0.05). At day 7 after ASCI, Rho A and ROCKII expression level in rats in TIIA group significantly increased compared with Fasudil group(P<0.05).3.3 At day 1 after ASCI, Rho A m RNA and ROCKII m RNA expression in rats in ASCI group significantly reduced compared with others(P<0.05). Rho A m RNA and ROCKII m RNA expression in rats in Fasudil group decreased significantly compared with TIIA group(P<0.05). In the mechanical damage of spinal cord neurons, Rho A m RNA and ROCKII m RNA expression in neurons in ASCI group were significantly higher than other groups(P<0.05). Rho A m RNA and ROCKII m RNA expression in neurons in Fasudil group was lower than those in TIIA group, but there was no significant difference(P>0.05).3.4 In vitro, the amount of MLC expression in neurons in ASCI group was no significant difference with other groups(P>0.05), while p-MLC expression quantity were significantly higher than other groups(P<0.05); p-MLC expression in neurons in TIIA group was lower than in Fasudil group, but there was no significant difference(p>0.05); Within each spinal cord neurons, p-MLC/MLC ratio were significantly higher in ASCI group than other groups(p<0.05); In vivo, at day 1 after ASCI, MLC expression in rats in ASCI group have no significant difference with other groups(P>0.05), and p-MLC expression quantity were significantly higher than other groups(P<0.05); p-MLC expression in TIIA group was lower than those in Fasudil group, but there was no significant difference(p>0.05);The ratio of p-MLC/MLC in rats and neurons in ASCI group were significantly higher than other groups(p<0.05); p-MLC/MLC ratio in rats and neurons in TIIA group was lower than those in Fasudil group, but not significantly(P>0.05).At day 7 after ASCI, MLC expression in rats in ASCI group had no significant difference with in the other groups(P>0.05), while p-MLC expression quantity were significantly higher than other groups(P<0.05); p-MLC significantly increased in rats in TIIA group than Fasudil group(P<0.05). P-MLC/MLC ratio in ASCI group were significantly higher than other groups(p<0.05); P-MLC/MLC ratio significantly increased in TIIA group compared with Fasudil group(p<0.05). ConclusionAccording to the results of immunohistochemical identification, we get the astrocytes and neurons layered co-culture system, and used for further study.TIIA plays a role of protection for spinal cord tissue and the neurons after ASCI through its oxidation resistance, inhibiting the inflammatory related factor secretion and antiapoptotic pharmacological effects similar to MP. TIIA combination with low dose MP therapy can reduce the useage of MP after ASCI, and can obtain similar nerve protective effect.Rho A/ROCKII/MLC signaling pathways is activated after ASCI, and inhibits axon regeneration process. TIIA can block the Rho A/ROCKII/MLC signal pathway after ASCI, reduces the expression of Rho A and ROCKII, inhibits the process of MLC phosphorylation, reduces the formation of p-MLC, and promotes neural axon regeneration and recovery of spinal cord function after ASCI.