| BackgroundCervical Spondylotic Myelopathy(CSM)is mainly manifested by spinal nerve dysfunction.It is a chronic spinal cord injury caused by multiple factors compressing the spinal cord.The initial symptoms of the disease are often difficult to detect,and the clinical symptoms are severe and the disability rate is high.In recent years,its incidence in the population has shown a significant increase,and the age of onset has also tended to be younger,which has caused great harm to the health and quality of life of middle-aged and elderly friends and even young people.Early surgical intervention in severe CSM patients has become an industry consensus,while for mild to moderate patients,conservative treatment should be considered first.Active intervention for CSM patients at the time of diagnosis can effectively delay the progression of the disease and protect the spinal cord function.However,the recognized treatment drugs for CSM are relatively scarce.Traditional Chinese medicine plays a role in protecting nerves and has been used in clinical practice for a long time.Among them,the Yishen Yangsui decoction used in this study is the effective experience formula of Zhu Liguo academician of the International Eurasian Academy of Sciences.It has obtained a patent.The previous retrospective analysis of the decoction for the treatment of 35 cases of mild to moderate myelopathy Cervical spondylosis patients have good curative effect.Subsequently,the research group conducted a multi-center randomized double-blind clinical study under the funding of the State Administration of Traditional Chinese Medicine’s special project of Chinese medicine industry scientific research "Research on the Prevention and Treatment of Cervical Spondylotic Myelopathy Comprehensive Program of Chinese Medicine".The clinical effective rate of Yishen Yangsui decoction in the treatment of cervical spondylotic myelopathy is 71.64%,which can significantly improve the sensory and motor functions of limbs in patients with cervical spondylotic myelopathy,which further confirms the exact curative effect of the compound.However,this prescription still lacks in-depth basic research evidence,which limits its clinical application to a large extent.Spinal cord injury leads to local tissue ischemia and hypoxia,abnormal inflammatory response and astrocyte proliferation,resulting in nerve regeneration disorders,spinal cord neuron death and corresponding functional loss.In the research of spinal cord injury regeneration and repair,the glial scar formed by the excessive proliferation of astrocytes has always been one of the important research directions.The glial scars formed by the damaged astrocytes in the early stage can form a barrier to prevent normal nerve tissue from being affected by inflammation and so on.After astrocytes mature,various functions beneficial to the body gradually decline or disappear.Studies have found that the existence of mature astrocytes or astrocyte scars is closely related to the failure of neuron axon regeneration.This experiment will carry out research from the perspective of the excessive proliferation of astrocytes affecting neuronal axon regeneration,enrich the scientific connotation of Yishen Yangsui decoction in the treatment of cervical spondylotic myelopathy,and provide a scientific basis for the new drug development and clinical application of this decoction.Research ContentObjective:To investigate the effect of Yishen Yangsui decoction on motor function,spinal cord tissue morphology of compressed segment,excessive proliferation of astrocytes,neuronal axon regeneration and myelination in rats with cervical spondylotic myelopathy.Methods:A total of 66 SPF-grade female SD rats(240±20)g were selected,and after 3 days of adaptive feeding,the baseline evaluation of spinal cord motor function was performed according to the BBB score,and the extreme value rats were excluded,and then randomly divided into 3 groups,blank control 8 rats in the group,8 rats in the sham operation group,and 50 rats in the model group.The CSM model was replicated by the method of compressing the spinal cord with the water-absorbing swelling material polyvinyl acrylamide interpenetrating network hydrogel.The BBB score and nuclear magnetic resonance were used 2 weeks after the modeling.Images were used to evaluate the model,and the extreme value rats were excluded,and then the model group was randomly divided into 5 groups,namely the low-dose,medium-dose,and high-dose groups of Yishen Yangsui decoction,the model control group,and the positive control group.According to the groups,they were given corresponding drug intervention for 4 weeks,and the rest were given an equal volume of distilled water.The motor function of the rats was evaluated before the intervention and 1,3,7,14,21,and 28 days after the intervention.The axon regeneration and myelination of spinal cord neurons were observed by transmission electron microscope.Result:(1)BBB score results:Two weeks after the operation,the spinal cord function of the rats in the blank control group and the sham-operated group was normal,and the difference was not statistically significant(P>0.05).At each time point after modeling,the BBB scores of limb motor function of the rats in the model group,the low-medium-high-dose Chinese medicine group,and the monosialotetrahexosylganglioside sodium group were significantly lower than those in the blank control group and the sham-operated group,and the difference was significant.There was statistical significance(P<0.05).After 7 days of intragastric administration,the motor function BBB score of the rats in the low,middle and high doses of Chinese medicine group and the monosialotetrahexose ganglioside sodium group was higher than that in the model group,and there was a statistical difference(P<0.05).(2)HE staining results:6 weeks after the operation,that is,4 weeks after administration,the white matter and gray matter of the spinal cord in the blank control group and the sham-operated group were intact;in the model group,neurons in the spinal cord were swollen and necrotic,the arrangement of nerve fibers was unclear,and the arrangement and structure of the spinal cord were disordered.The white matter is loose,with a large number of cavities,axonal rupture and demyelination changes;Compared with the model group,the structure of spinal cord tissue in each Chinese medicine group and positive control group was clearer,the shape of nerve cells tended to be normal,and the pyknosis,dissolution and necrosis of neurons were alleviated.(3)Real time-PCR test results①4 weeks after administration,there was no significant difference in the expression of GFAP mRNA in the spinal cord tissue of the sham-operated group(P>0.05).Compared with the model group,the expression of GFAP mRNA in the spinal cord tissue of the rats in each administration group was attenuated(P<0.05).②6 weeks after the operation,that is,4 weeks after administration,there was no significant difference in the expression of GAP-43 mRNA in the spinal cord tissue of the rats in the sham operation group(P>0.05).The expression of GAP-43 mRNA was enhanced(P>0.05);the expression of GAP-43 mRNA in the spinal cord tissue of the rats in each administration group was stronger than that in the model group(P<0.05).(4)Western blot detection resultsAfter the intervention,there was no significant difference in the expression of GAP-43 in the spinal cord tissue of the blank control group and the sham operation group(P>0.05);the expression of GAP-43 in the spinal cord tissue of the model group was higher than that of the blank control group and the sham operation group,but there was no statistical difference(P>0.05);compared with the model group,the expression of GAP-43 in the spinal cord tissue of the rats in the low and medium doses of Yishen Yangsui decoction group increased,and the difference was statistically significant(P<0.05).(5)Immunofluorescence staining resultsAfter the intervention,there was no significant difference in the GFAP fluorescence intensity between the blank control group and the sham-operated group(P>0.05);the GFAP fluorescence intensity of the blank control group and the sham-operated group was lower than that of the model group,and there was a statistical difference(P<0.05);Compared with the model group,the GFAP fluorescence intensity in the spinal cord tissue of the rats in the low-medium-high-dose Yishen Yangsui decoction group and the positive control group decreased(P<0.05).Among them,the expression of GFAP fluorescence intensity was the lowest in the middle-dose group of Yishen Yangsui decoction.(6)TEM results6 weeks after the operation,that is,4 weeks after administration,the axons and myelin sheaths in the spinal cord tissues of the rats in the blank control group and the sham-operated group showed normal structures;the myelin sheaths in the model group showed "onion skin"-like release,loss,and rupture,axonal degeneration and atrophy;compared with the model group,the demyelination and axonal degeneration of each administration group were improved to varying degrees.Conclusion:(1)Yishen Yangsui decoction can promote the recovery of spinal cord motor function in CSM rats;(2)Yishen Yangsui decoction can improve the histomorphological changes of the compressed spinal cord in CSM rats;(3)Yishen Yangsui decoction can reduce the overexpression of GFAP in the compressed spinal cord of CSM rats and inhibit the excessive proliferation of astrocytes;(4)Yishen Yangsui decoction can up-regulate the expression of GAP-43 in the compressed spinal cord of CSM rats,and promote neuronal axon regeneration;(5)The mechanism of action of Yishen Yangsui decoction in the treatment of CSM may be related to inhibiting the excessive proliferation of astrocytes and reducing the hindrance of glial scar on neuronal axon regeneration.Innovation point:Taking the excessive proliferation of astrocytes affecting neuronal axon regeneration as the breakthrough point,the neural repair mechanism of Yishen Yangsui decoction on chronic spinal cord injury is discussed from the aspects of animals,tissues and molecules;Reveal the scientific connotation of traditional Chinese medicine compound in the treatment of CSM. |
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