| Due to the difficulty to repair the central nervous system(CNS),patients often have different degrees of motor neurological dysfunctions and sensory neurological dysfunctions left over after ischemic stroke,which severely affect the normal life of patients and their families.As the only clinically recognized effective therapy for stroke,the use of tissue plasminogen activator(t PA)is severely restricted because of the narrow therapeutic window and side effects.Therefore,finding effective drugs which can promote nerve repair and improve quality of life has become a problem urgently needed to be solved.In recent decades,studies have shown that the CNS of adult mammals shows self-repair.The effective regeneration of axons and the reconstruction of connections with peripheral nervous tissues play a vital role in nerve recovery.However,due to the existence of a large number of axonal regeneration inhibitors in the microenvironment in the injured sections,axons cannot effectively regenerate and nerves are difficult to repair.Nogo-A is the most critical axonal regeneration inhibitor.It binds to the receptor to trigger the downstream Rho A/ROCKII signaling pathway.Activated ROCKII promotes the phosphorylation of the target protein myosin light chain(MLC),which in turn induces cytoskeletal rearrangement,growth cone collapse and ultimately impedes axonal regeneration.It can be confirmed that Nogo-A/Ng R1/Rho A/ROCKⅡ/MLC signaling pathway plays a key negative regulatory role in nerve repair.Therefore,drugs targeted to inhibit the expression of Nogo-A and its signaling pathway are expected to be used in the treatment of ischemic stroke.Ischemic stroke belongs to the category of "stroke" in traditional Chinese medicine.The pathogenesis is mostly Qi deficiency and blood stasis,and the treatment is mainly based on invigorating Qi and promoting blood circulation.We consider that the basic pathogenesis of this disease is "deficiency of Qi and blood with blood stasis".Treatment should be focused on nourishing blood on the basis of invigorating Qi and promoting blood circulation,with a view to achieving the purposes of invigorating Qi and blood,and nourishing blood and promoting blood circulation.Hexue Shengluo Recipe(HXSL)was based on Renshen Yangrong Decoction and had achieved good effects in the clinic.Previous studies have shown that Hexue Shengluo Recipe has good neuroprotective effects on ischemic stroke,which is related to promoting angiogenesis and nerve regeneration after cerebral ischemia.However,the effect of TSA on axonal regeneration following cerebral ischemia and its mechanism remain unknown,which are the purposes of this study.In this study,three-part animal experiments were conducted to investigate whether Hexue Shengluo Recipe promoted axonal regeneration in focal cerebral ischemia in rats by inhibiting the Nogo-A/Ng R1/Rho A/ROCKⅡ/MLC signaling pathway.Furthermore,the neuroprotective effects of Hexue Shengluo Recipe on focal cerebral ischemia in rats were verified.Part one Neuroprotective effects of Hexue Shengluo Recipe on focal cerebral ischemia in ratsObjective: Objective: To investigate the neuroprotective effects of Hexue Shengluo Recipe on focal cerebral ischemia in rats through the evaluations of m NSS,screen test,TTC staining,HE staining and Nissl staining in permanent middle cerebral artery occlusion(p MCAO)rats.Methods: Adult male Sprague-Dawley(SD)rats weighing 250-280 g,were selected to prepare a permanent middle cerebral artery occlusion(p MCAO)model by using the suture method.Rats were divided randomly into 6 groups.A sham operation group(Sham): rats underwent the same surgery without the insertion of a suture and were administered an equal amount of normal saline;a sham operation+HXSL group(Sham+HXSL): rats underwent the same surgery without the insertion of a suture and were administered 15.3 g/kg HXSL;a vehicle group: rats received p MCAO and were administered an equal volume of normal saline;a HXSL group(HXSL):rats received p MCAO and were administered 15.3 g/kg HXSL;a Fasudil group(Fasudil): rats received p MCAO and were administered 10 mg/kg FH;a Tanshinone IIA group(TSA): rats received p MCAO and were administered 20mg/kg TSA.Rats in each group were assessed for m NSS scoring,screen test and general state observation before modeling and on the 3rd,7th,14 th,21st and 28 th days after modeling.TTC staining was used to measure cerebral infarct volume on the 7th day after modeling.HE staining and Nissl staining were performed to observe the morphological changes of brain tissue and measure the number of intact neurons on the 7th and 14 th days after modeling.Results:1.After modeling,rats in the Sham group and Sham + HXSL group quickly recovered quickly as usual;rats in each p MCAO group were in a state of mental depression,unresponsiveness and curling up,with the dark and dry fur and the decreased food and defecation.Compared with the Vehicle group,rats in the HXSL group,Fasudil group,and TSA group all improved significantly in terms of mental state,flexibility,intake,defecation,and body weight.2.The results of m NSS scoring system showed that on the 3rd,7th,14 th,21st and 28 th days after modeling,rats in the Sham group and Sham + HXSL group scored 0 and did not show any functional impairments.Compared with the Sham group,the Vehicle group showed the significantly raising scores(P< 0.01).Compared with the Vehicle group in the same period,the m NSS scores of the HXSL group,Fasudil group,and TSA group were significantly reduced(P < 0.01).3.The results of screen test showed that in each time period after the modeling,rats in the Sham group and Sham + HXSL group scored 0 and did not show any abnormal changes in the muscle strength.Compared with the Sham group,the Vehicle group showed the significantly raising scores(P <0.01).Compared with the Vehicle group,the scores of the HXSL-M group were significantly reduced from the 7th day after modeling(P < 0.01),while the scores of the Fasudil group and TSA group were significantly reduced from the 14 th day after modeling(P < 0.05 or P < 0.01).The results of the screen test were consistent with the m NSS.4.The results of TTC staining showed that on the 7th day after modeling,there were no cerebral infarcts in rats in the Sham group and Sham + HXSL group.Compared with the Sham group,the cerebral infarct volume of rats in the Vehicle group was significantly increased(P < 0.01).Compared with the Vehicle group,the cerebral infarct volume of rats in the HXSL group,Fasudil group and TSA group was significantly reduced(P < 0.01).5.The results of HE staining showed that on the 7th and 14 th days after modeling,the cell structure and morphology of the cerebral cortex in rats in the Sham group and Sham+HXSL group were normal,and there was no significant difference between the two groups.Compared with the Sham group,the cell structure and morphology of the cerebral cortex in rats in the the Vehicle group were destroyed,with significant increased denatured cells.Compared with the vehicle group,treatment of HXSL,Fasudil and TSA all improved the brain tissue morphology and ameliorated the cell damage.6.The results of Nissl staining showed that on the 7th and 14 th days after modeling,cells in the Sham group and Sham+HXSL group were were arranged regularly,with the complete structure.There was no significant difference in the number of neurons between the two groups(P > 0.05).Compared with the Sham group,the nerve cells in the Vehicle group were loosely arranged with the fuzzy structure,and the number of neurons was significantly reduced(P < 0.01).Compared with the Vehicle group in the same period,the morphology and structure of nerve cells in the HXSL group,Fasudil group,and TSA group were significantly improved,and the number of neurons was significantly increased(P < 0.01).Summary:1.In this experiment,the p MCAO model was successfully established by the suture method.It was confirmed that Hexue Shengluo Recipe significantly reduced the neurological deficits and promoted the recovery of neurological function in p MCAO rats.2.Hexue Shengluo Recipe significantly diminished cerebral infarct volume and relieved the pathological damage in the cortex surrounding the infarct in p MCAO rats,thus showing a significant positive neuroprotective effect.Part two Effects of Hexue Shengluo Recipe on Axonal Regeneration in Focal Cerebral Ischemia in RatsObjective: To investigate the effects of Hexue Shengluo Recipe on axonal regeneration in cerebral cortex in p MCAO rats by observing the growth of axons and measuring the axonal length and the expression of axonal regeneration-related markers NF200 and GAP-43 in the cerebral cortex in p MCAO rats.Methods: Adult male Sprague-Dawley(SD)rats weighing 250-280 g,were selected to prepare a permanent middle cerebral artery occlusion(p MCAO)model by using the suture method.Rats were divided randomly into 6 groups: a sham operation group(Sham),a sham operation+HXSL group(Sham+HXSL),a vehicle group(Vehicle),a HXSL group(HXSL),a fasudil group(Fasudil)and a Tanshinone IIA group(TSA).On the 7th,14 th and 28 th days after modeling,rats in each group were observed for axonal morphology,growth,and expression of NF200 by immunofluorescence staining;rats were measured for the expression of GAP-43 by Western blot and the m RNA levels of GAP-43 and NF200 by real-time quantitative PCR(q RT-PCR).Results:1.The results of immunofluorescence staining showed that on the 7th,14 th and 28 th days after modeling,the morphology of the Sham group and Sham+HXSL group was similar,and there was no significant difference in axonal length between two groups(P > 0.05).Compared with the Sham group,the axonal structure of the Vehicle group was fragmented and the length was significantly reduced(P < 0.01).Compared with the Vehicle group in the same period,the morphology and structure of axons in the HXSL group,Fasudil group,and TSA group were significantly improved,and the length was significantly increased(P < 0.01).2.Protein and m RNA levels of NF200 in the cortex surrounding the infarct.The results of immunofluorescence staining showed that on the 7th,14 th and 28 th days after modeling,the expression of NF200 in the cerebral cortex of the Sham+HXSL group and Sham group was similar(P > 0.05).Compared with the Sham group in the same period,the expression of NF200 protein in the Vehicle group was significantly reduced(P < 0.01);compared with Vehicle group,the expression of NF200 in the HXSL group,Fasudil group and TSA group were significantly increased(P < 0.05 or P < 0.01).The results of q RT-PCR showed that on the 7th,14 th,and 28 th days after modeling,the m RNA levels of NF200 in the cerebral cortex in the Sham+HXSL group and the Sham group were similar(P > 0.05);compared with the Sham group,the m RNA levels of NF200 in the Vehicle group were significantly reduced(P < 0.01);compared with Vehicle group,the m RNA levels of NF200 in the HXSL group,Fasudil group,and TSA group were significantly increased(P < 0.01).3.Protein and m RNA levels of GAP-43 in the cortex surrounding the infarct.The results of Western blot showed that on the 7th,14 th,and 28 th days after modeling,the expression of GAP-43 in the cerebral cortex in the Sham+HXSL group and the Sham group was similar(P > 0.05);compared with the Sham group in the same period,the expression of GAP-43 in the Vehicle group was increased(P < 0.01);compared with Vehicle group,the expression of GAP-43 in the HXSL group,Fasudil group and TSA group were significantly increased(P < 0.01).The results of q RT-PCR showed that on the 7th,14 th,and 28 th days after modeling,the m RNA levels of GAP-43 in the cerebral cortex in the Sham+HXSL group and the Sham group were similar(P > 0.05);compared with the Sham group,the m RNA levels of GAP-43 in the Vehicle group were increased(P < 0.01);compared with Vehicle group,the m RNA levels of GAP-43 in the HXSL group,Fasudil group,and TSA group were significantly increased(P < 0.01).Summary:1.Hexue Shengluo Recipe improved axonal morphology and promoted axonal growth and extension in the cortex surrounding the infarct in p MCAO rats.2.Hexue Shengluo Recipe significantly enhanced the protein and m RNA expression of NF200 and GAP-43 in the cortex surrounding the infarct in p MCAO rats,thus significantly promoting axonal regeneration after cerebral ischemia.Part three Effects of Hexue Shengluo Recipe on Nogo-A/Ng R1/Rho A/ROCKII/MLC Signaling Pathway in Focal Cerebral Ischemia in RatsObjective: To investigate the effects of Hexue Shengluo Recipe on Nogo-A/Ng R1/Rho A/ROCKII/MLC signaling pathway in p MCAO rats by quantifying the protein and m RNA expression of Nogo-A,Ng R1,Rho A,ROCKⅡ,MLC and p-MLC in the cortex surrounding the infarct in p MCAO rats.Methods:Adult male Sprague-Dawley(SD)rats weighing 250-280 g,were selected to prepare a permanent middle cerebral artery occlusion(p MCAO)model by using the suture method.Rats were divided randomly into 6 groups: a sham operation group(Sham),a sham operation+HXSL group(Sham+HXSL),a vehicle group(Vehicle),a HXSL group(HXSL),a fasudil group(Fasudil)and a Tanshinone IIA group(TSA).On the 7th,14 th and 28 th days after modeling,rats in each group were measured for the protein and m RNA expression of Nogo-A,Ng R1,Rho A,ROCKⅡ,MLC and p-MLC by western blot and q RT-PCR.Results:1.The results of Western blot showed that on the 7th,14 th,and 28 th days after modeling,the protein expression of Nogo-A,Ng R1,Rho A,ROCKⅡ and p-MLC in the cerebral cortex in the Sham+HXSL group and Sham group were similar,thus there was no significant difference between two groups(P > 0.05).Compared with the Sham group during the same period,the protein levels of Nogo-A,Ng R1,Rho A,ROCKⅡ,and p-MLC in the Vehicle group were significantly increased(P < 0.01);compared with the Vehicle group,the protein levels of Nogo-A,Ng R1,Rho A,ROCKⅡ,and p-MLC in the the HXSL group,Fasudil group,and TSA group were significantly down-regulated(P < 0.05 or P < 0.01).However,there was no significant difference in MLC protein expression in rats in each group during all periods after modeling(P > 0.05).2.The results of q RT-PCR showed that on the 7th,14 th,and 28 th days after modeling,the m RNA levels of Nogo-A,Ng R1,Rho A,and ROCKⅡ in the cerebral cortex in the Sham+HXSL group and Sham group were similar,thus there was no significant difference between two groups(P > 0.05).Compared with the Sham group in the same period,the m RNA levels of Nogo-A,Ng R1,Rho A,and ROCKⅡ in the Vehicle group were increased(P <0.01);compared with the Vehicle group,the m RNA levels of Nogo-A,Ng R1,Rho A,and ROCKⅡ in the HXSL group,Fasudil group,and TSA group were significantly reduced(P < 0.01).However,there was no significant difference in MLC m RNA expression in rats in each group during all periods after modeling(P > 0.05).Summary:1.Hexue Shengluo Recipe significantly down-regulated the expression of protein and m RNA of Nogo-A,Ng R1,Rho A,ROCKⅡ and p-MLC in the cortex surrounding the infarct in p MCAO rats,thus inhibiting the activation of this signaling pathway.2.Combined with the results above mentioned,it was suggested that by inhibiting the activation of Nogo-A/Ng R1/Rho A/ROCKⅡ/MLC signaling pathway,Hexue Shengluo Recipe promoted axonal regeneration in the cortex surrounding the infarct in p MCAO rats,thus playing a positive neuroprotective role in p MCAO rats.Conclusions:1.In this experiment,the p MCAO model was successfully established by the suture method.Hexue Shengluo Recipe significantly improved the symptoms of neurological deficits,reduced the volume of cerebral infarct,and relieved neuronal damage in the cortex surrounding the infarct.Therefore,Hexue Shengluo Recipe had a good neuroprotective effect on p MCAO rats.2.Hexue Shengluo Recipe promoted axonal regeneration in the cortex surrounding the infarct in p MCAO rats.3.The role of Hexue Shengluo Recipe in promoting axonal regeneration in the cortex surrounding the infarct in p MCAO rats and improving nerve function recovery may be related to down-regulating the expression of Nogo-A/Ng R1/Rho A/ROCKⅡ/MLC signaling pathway. |
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