| ObjectiveAmyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neuron degenerative disease that major involve the motor neuron. There is no effective treatments currently.This study proposed by different concentrations of Jianpi yifei Recipe animal models for the treatment of ALS hSOD1-G93A transgenic mice, by detecting disease phenotype and spinal cord anterior horn motor neurons survival situation to evaluate the effect of Jianpi yifei Recipe. Through the detection of the spinal cord malondialdehyde, such as superoxide dismutase (SOD) activity to evaluate oxidation of free radicals function of Jianpi yifei Recipe, formula for traditional Chinese medicine treatment of ALS curative effect and mechanism of animal models provide basic material.Methods1 Reagents and equipments:1.1 The Jianpi yifei Recipe:Astragalus membranaceus 9 g, Codonopsis pilosula 4.5 g and 4.5 g Atractylodes macrocephala,1 g Poria cocos, Ophiopogon japonicus 3 g and 0.5 g Citrus reticulata Blanco, Pinellia ternata of 0.5 g and 1 g Belamcanda chinensis 1 g,0.5 g Acorus tatarinowii, Typhonium giganteum Engl 0.5 g, Beauveria bassiana 0.5 g and 0.1 g Strychnos nux-vomica L, Glycyrrhiza uralensis Fisch 2 g. Purchase all particles form, unified model for the same batch, bought from Jiang Yin Tian Jiang company.1.2 MDA, GSH, SOD and GSH-Px detection kits were purchased from Nanjing Jian Cheng institute of biological engineering; Tar bought a purple from Sigma-Aldrich company, other common reagents for the analysis pure from domestic.2 Experimental animals and grouping:2.1 hSODl-buy G93A transgenic mice from NJU institute of biology.2.2 Animal grouping:8 weeks mice, lavage daily, which is divided into five groups,8 weeks of hSODl G93A transgenic mice-a total of 24, six in each group, according to the number were randomly divided into four groups, (1) physiological saline group (2) low dose of traditional Chinese medicine group (2.86 g/Kg) and (3) middle dose of Chinese traditional medicine group (5.72 g/Kg) (4) high dose of Chinese traditional medicine group (11.44 g/Kg). Eight weeks of common wild type mice, a total of 6 for normal control group, physiological saline infusion. Observe the movement function and survival of mice,16 weeks between groups rats were taken each rat age 3, only on the spinal cord anterior horn motor neurons, surname dyeing, the other 3 in each group only for oxidation of free radicals and immunofluorescence test.3 Steps:3.1 The weight change of the model miceUse electronic weighing the weight of model mice every day, and make records3.2 The Jianpi yifei Recipe disease phenotype after formula for evaluation:3.2.1 Onset time (daily):four points Weydt method to assess:four points: no movement dysfunction;Three points:in the time of tail suspension hind legs trembling;Two points:the abnormal gait;One point:at least one hind leg appear drag;Zero point:the rat body flip for supine within 30s do not turn for the prone position. The onset of evaluation:a retrospective analysis (＞2 weeks), to score＜4 the first time as the onset time.3.2.2 Survival:the time of death:the body of the rat flip for supine within 30s do not turn for prone position that score of 0 point, recorded as death.3.2.3 Movement function:3.2.3.1 Tail suspension test:suspension rat tail, observe the stretching and bending of the hind legs, and to score. Two points:normal; one point:partly normal;Zero:missing.3.2.3.2 Traction test:put the mice on the 20cm long wire, the number of mice falling within 5 min (mice after falling, will continue to test on its back on the wire again).3.2.3.3 Run test:put the mice in particular turn bar apparatus, in order to speed the 16r/min lasts for 5 min, record the type of average drop bar number (mice after drop bar, turn its back on the run again to continue the test on the rod).3.3 Jianpi yifei Recipe formula pathological changes after treatment:the application of 4%paraformaldehyde after cardiac perfusion, fixed after 24 hours, dehydration, sucrose gradient using OCT embedding, take lumbar segment of the spinal cord were frozen section (20um slice thickness), every three piece take 1 piece. Application of tar purple’s staining. Anterior horn motor neurons counts standard:Nissl body structure with clear, clear nuclei and nucleoli, cell body larger polygonal cells.3.4 The treatment mechanism of Jianpi yifei Recipe:Oxidation of free radicals, glucosinolates barbituric acid method for determining the content of MDA;The activity of SOD, nitrite method, Disulfide generation two nitro benzoic acid method for determining the content of GSH;Two nitro benzoic acid method for determining the activity of GSH-Px, according to the kit instruction manual operation.Results1. Weight8 weeks of age began to determine weight in mice, before 12 weeks weight there was no significant difference between groups, since 13 weeks weight increased in high dose Jianpi yifei Recipe group than other group, the difference is significant (P<0.05). About 14 weeks, weight decline model mice, 14-20 weeks of low dose group, middle dose and high dose are lower than the normal saline group, body weight loss has a significant difference (P＜0.01).2. The animal survival and onset timeThe influence of normal saline group model nice in clinical symptoms (101.5 ±3.45) days, namely Weydt four points method, score 3 points (period), a hind leg weakness, tail suspension after hind legs tremble, and weakness, to end-stage bilateral hind leg unbend sample stiffness, a carpenter, eventually died because of difficulty in feeding and breathing failure, the average survival time was (133.2±2.32) days. And low dose, middle dose and high dose Jianpi yifei Recipe time of treatment group were (103.3±2.16) days, (109.5 ±1.87) days and (112±3.03) days, the difference is significant with middle dose and high dose of Jianpi yifei Recipe treatment group. Low dose, middle dose and high dose of Jianpi yifei Recipe treatment group survival, respectively, (134.3±1.86) days, (140.2±2.32) days, (147.8±1.17) days. While middle dose and the high dose treatment group prolong survival time than normal saline group and low dose treatment groups, the difference is significant (P＜0.01).3. The movement functionTraction test:at the same time, different concentrations of Jianpi yifei Recipe are not the same, high dose of Jianpi yifei Recipe is more better than the low and the middle dose treatment groups in tolerance, clear difference compared with normal saline group (P＜0.01).Run test:in the same period, the high dose of Jianpi yifei Recipe is less fall than the middle dose treatment groups, clear difference compare with the low dose of Jianpi yifei Recipe and saline group (P＜0.01).4. Spinal cord anterior horn motor neurons countsWith the increase of age of rats, mice model of lumbar segment the neurons of the anterior horn of spinal cord lesions has emerged, austenite is not clear, modified nucleus pycnosis, cell volume decreases, and cells atrophy, decreased, the whole spinal cord ventral horn shape atrophy, disease progression with time.18 weeks each pathological’s dyeing as shown in figure 1. Saline treatment group motor neuron count was (3.86±1.215)/field, low, middle and high dose Jianpi yifei Recipe group were (4.00±0.816)/field, (4.57±0.976)/field and (7.57±0.976)/field, high dose of Jianpi yifei Recipe is compared between treatment groups and other groups have significant difference (P< 0.01).5. Antioxidant glutathione (GSH) content of free radicals and glutathione peroxidase activity (GSH-Px), SOD and MDAPhysiological saline group of GSH, GSH-Px and SOD activity value of the minimum, MDA activity value of the maximum and Jianpi yifei Recipe group of GSH, GSH-Px and SOD activity increased with the increased concentration of Jianpi yifei Recipe, MDA on the contrary, comparison between groups have significant difference.(P< 0.01)ConelusionJianpi yifei Recipe can significantly improve model mice body weight, can prolong the onset time of mice model. And a dose of relevance. Observe the movement function of mice in the traction test can decrease the number of falls for mice. Surname dyeing waist marrow of anterior horn motor neurons was found Jianpi yifei Recipe treatment groups has more survival cells. Detection of spinal cord tissue by active, found that the activity of GSH, GSH-Px and SOD increased obviously, MDA on the contrary, Jianpi yifei Recipe may protect neurons by oxidation of free radicals.This research first applied classic model of ALS hSODl-G93A curative effect and mechanism of traditional Chinese medicine (TCM) study of transgenic mice for traditional Chinese medicine treatment of ALS research provides a new animal model. First applied different concentrations of Jianpi yifei Recipe monotherapy ALS, targets of Jianpi yifei Recipe root and pharmacological mechanism are discussed in this paper, provides a theoretical basis for the development of traditional Chinese medicine treatment of ALS. |
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