Neuroprotective Effect Of Jiweiling Injection On Motor Neuron Disease

The motor neuron disease (MND) refers to the chronic and degenerative diseases of selective injury of anterior horn of spinal cord, motor neuron of the brain stem and pyramidal tract, which mainly includes amyotrophic lateral sclerosis (ALS), progressive spinal myodystrophy (PSMA), progressive bulbar palsy(PBP) and primary lateral sclerosis(PLS). MND is characterized by the symptoms of myatrophy, weakness and muscular fibrillation and the bulboparalysis symptoms of dysarthria, dyspnea, dietary cough and short of breath. The cause and pathogenesy of this progressive disease is still unknown. No effective drug and method has been found to treat the disease in western medicine. It is necessary to seek the effective neruoprotectant to decrease the degeneration injury and delay the course of the MND and to look for the neuroprotection treatment project. Jiweiling injection(JWL), under the director of Qi-jing theory and Luo-bing doctrine, is successfully manufactured by professor Wu yi-ling to treat the motor neuron disease. JWL injection has the obvious curative effect in the clinical application and animal experiment. Objective The main objective of this study as follows: 1. Approach the traditional Chinese medicine pathogenesis of MND by the Qi-jing theory and Luo-bing doctrine so as to seek the effective method and drug of traditional Chinese medicine. 2. To observe the effects of traditional Chinese medicine JWL injection on the growth of primary cultured motor neurons of embryonic rat spinal cord as to provide evidence for the clinical application. 3. To observe the protective effects of traditional Chinese medicine JWL injection on the cultured motor neurons of embryonic rat spinal cord damnified by the excitatory amino acid (EAA) neurotoxicity and to approach the protective effect and its mechanism of traditional Chinese medicine. 4. To observe the effects of Jiweiling injection on neural stem cells(NSCs) proliferation and differentiation of embryonic rat and to approach the mechanism of the traditional Chinese medicine method Fuyuanqiwei. 5. To observe the effects of Jiweiling injection on human bone marrow stromal cells (BMSCs) proliferation and differentiation and on embryonic neural stem cells differentiation with the cell-conditioned medium (CM) as to provide experimental evidence for treating MND by traditional Chinese medicine associated with stem cell transplant. Methods 1. To sum up the past dynasties traditional Chinese medicine documents of Weizheng combing with the modern medicine development of motor neuron disease, lucubrate the traditional Chinese medicine mechanism of MND by the the Qi-jing theory and Luo-bing doctrine. 2. Spinal motor neurons (SMN) were separated from embryonic rat spinal cord by density gradient centrifugation and primary cultured. Experimental groups were treated by JWL of different concentration diluting with fresh culture medium and riluzole group was treated by riluzole(Final concentration was 10μmol?L^-1). The cell vigor of cultured motor neurons was tested by 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and the neurites outgrowth of motor neurons by neurofilament-200(NF-200) immunohistrochemical staining and by means of computer image analysis system to measure the lengths of nervous process. 3. Motor neurons were separated from embryonic rat spinal cord by density gradient centrifugation and primary cultured 3days. Experimental groups were treated by JWL of different concentration diluting with fresh culture medium(0.5%,1%,5%JWL) and riluzole group was treated by riluzole(Final concentration was 10μmol.L^-1). Motor neurons were treated with the final concentration 0.05mmol.L^-1 glutamate(Glu) to result in the the excitatory amino acid (EAA) neurotoxicity trauma to evaluate theprotective effect of the JWL injection. The cell vigor of cultured motor neurons was tested by MTT method, the lactic dehydrogenase (LDH) vigor of culture liquid were tested by the automatic biochemistry analysis machine and apoptosis by the terminal-deoxynucleotidy transferase mediated nick end labeling (TUNEL ) positive neurons counts. The change of Ca2+ concentration in the cell was tested by the fluorescence probe label technique and the laser scanning confocal microscopy (LSCM). 4. Neural stem cells (NSCs) were isolated and cultured from rat embryoes under integral asepsis condition and induced to differentiate for 7 days by different concentration of JWL injection diluting by fresh medium. The proliferation of the NSCs was tested by the liquid scintillation counting after adulterating 3^H-Thymidine (3^H-TdR) . Flow-cytometer technique and immunohistochemical staining method were used to test the expression of neurofilament-200 (NF-200) and glial fibrillary acidic protein (GFAP) as to study the differentiation of NSCs. 5. The human marrow stromal cells(BMSCs) were isolated by density gradient centrifugation and primary cultured. Experimental groups were treated by JWL of different concentration diluting with fresh culture medium(0.1%,0.2%,0.5%,1%,2%,5%JWL) and control group was treated by fresh medium. The proliferation of the BMSCs was tested by the liquid scintillation counting after adulterating 3^H-Thymidine(3^H-TdR). Flow-cytometer technique and immunohistochemical staining were used to test the expression of neurofilament-200 (NF-200) and glial fibrillary acidic protein (GFAP) after the differentiation of BMSCs. The neuron stem cells(NSCs) were isolated from human fetal brain and purifed by transfer of culture under integral asepsis condition. The cell-conditioned medium (CM) was collected from the BMSCs and was affiliated into the NSCs as the CM control group. Experimental groups were treated by different concentration JWL injection(0.1%,0.2%,0.5%,1%,2%,5%JWL) at the same time. The differentiation was test by the flow-cytometer technique and immunohistochemical staining.Results 1. Motor neuron disease belonged to the Weizheng category according to its clinical symptoms. Motor neuron disease located in the encephala and belonged to Qijing-luo disease. The deficit and deficiency of Qi-jing was the fundamental TCM mechanism of motor neuron disease and the weakness and stagnant of Luoqi was the important pathologic tache. JWL injection had the function of Fuyuanqiwei, Yangrongshenji, and Yiqitongluo and its prescription was brief and precise. 2. The motor neurons of control group grew slowly. The MN in JWL injection group grew better than that in control and the differences of the cell vigor between these groups were significan(tP<0.05). The optic density (OD) value in 1%JWL group was much higher than that in riluzole group(P<0.05). And there was no difference of OD value between riluzole group and control group. The leading neurite of motor neuron was long and symmetrical and there was no filiation or at the distal. There was no difference of the leading neurite length between control and riluzole group(p>0.05). The neurite in 0.5%JWL and 1%JWL group grew faster and the differences of the leading neurite length between JWL group and control group were significan(tp<0.05). There was no obvious effect of 5% JWL on the neurite outgrowth of spinal motor neurons and there was no difference comparing control group (p>0.05). 3.The cell vigor of the spinal motor neuron descended distinctly after damaged by glutamate 24 hours and the OD value by MTT assay was about one tenth of that of the normal motor neurons. The OD values in 1%JWL and riluzole group were higher than that in control group(p<0.05) while there was no difference between 1% and riluzole group(p>0.05). There was no diffecnece of the OD value in 0.5%JWL, 1%JWL and control group(p>0.05)although there was hoist tendency of the OD value in 0.5%JWL group. The lactic dehydrogenase(LDH)of culture liquid in 0.5%JWL and riluzole group were both lower than that in control group tested by the automatic biochemistry analysis machine(p<0.05) and there was no differencebetween 0.5%JWL and riluzole group. It suggested that 0.5%JWL and riluzole could decrease the LDH leakage of the motor neuron damaged by glutamate. There was no difference of the LDH leakage between 1%JWL, 5%JWL and control group.Apoptosis was induced by the little dosage glutamate and apoptosis body appeared by terminal-deoxynucleotidy transferase mediated nick end labeling (TUNEL). TUNEL positive cell numbers in JWL and riluzole group were less than that in control group and the differences between the 1% JWL, riluzole group and control group(p<0.05). There was no difference between 1% JWL and riluzole group. The TUNEL positive cells in 0.5%JWL and 5%JWL group decreased but there was no difference comparing the control group(p>0.05). The fluorescein of the motor neuron in glutamate control group boosted up while there was no obvious change of the fluorescein in 1%JWL group under the laser scanning confocal microscopy(LSCM). It was observed from the curve that the calcium icon concentration in glutamate control group significantly increased while there was no obvious change of calcium icon concentration in 1%JWL group. It suggested that JWL could restrain the calcium overload in the motor neuron induced by the glutamate. 4. The results of NSCs by the liquid scintillation counting after adulterating 3H-Thymidine(3H-TdR) showed that the count per minute (CPM) in 0.1%,0.5%,1%JWL groups increased sharply and the difference comparing with that in control group was significant(p<0.01). There was no difference between 0.5% JWL and 1%JWL group(p>0.05) and the difference among the other groups was significant(p<0.01). it suggested that JWL injection could promote the proliferation of the NSCs and the function was relative to the concentration. The results tested by the flow-cytometer technique showed that the GFAP positive cells, the ratio of (50.86±3.42)%, was more than the NF-200 positive cells, the ratio of (37.53 ±2.46)% in control group after differentiation. The NF-200 positive cells increased obviously in the 0.1%,0.5%,1%JWL groups and the difference between the ratio of NF-200 positivecells, (37.53±2.46)% in 1%JWL group and that in control group was significant. However, the GFAP positive cells decreased and there was significant difference between 1%,5%JWL group and control group. 5. The human BMSCs grew better and proliferated faster in JWL groups and the CPM increased sharply by the the liquid scintillation counting after adulterating (3H-TdR) , the difference comparing the control group was significant(p<0.01). There were no difference between 0.1% JWL and 0.2% JWL group, between 0.2%,0.5%JWLgroup and 2% JWL group(p>0.05). The difference was significant among the other groups ( p<0.05 or p<0.01).The cell numbers that human BMSCs differentiated into neuron and gliocyte in control group after adding the serum culture medium were very slow which the ratios respectively were only(5.85±1.03)% and( 8.90±1.44)%. The NF-200 and GFAP positive cells increased obviously in all JWL groups, the difference was significant comparing the control group (p <0.01)and the function was relative to the concentration. Under the function of BMSCs cell-conditioned medium(CM), human NSCs in CM control group mainly differentiated into the gliocyte and the ratio of GFAP positive cells (63.38±5.72)% was more than neuron, the ratio of(25.24±4.61)% . The NF-200 positive cells increased along with the concentration of JWL injection increasing and the difference was significant comparing with that in CM control group(p<0.01).While The GFAP positive cells decreased along with the concentration of JWL injection increasing and the difference was significant comparing with that in CM control group(p<0.01). NSCs differentiation gave priority to NF-200 positive cells after the concentration of JWL injection in excess of 0.2% and the NF-200 positive cells exceeded the GFAP positive cell. Conclusion The theme include the theory discussion and experimetnal research of the neruroprotective effect of JWL injection on the motor neuron disease. The result suggested that motor neuron disease located in the encephala and belonged to Qijing-luo disease and its TCM pathological mechanism was...