| Objective:Myasthenia Gravis(MG)is mediated by antibodies to acetylcholine receptor(AChR)and participated by complements,mainly involving postsynaptic acetylcholine receptors of neuromuscular junctions.It is an acquired autoimmune disease that relies on cellular immunity.Females aged 20-30 are at high incidence and often accompanied by thymic hyperplasia;while males aged 40-50 are at high incidence and often accompanied by thymoma and other autoimmune diseases.In a word,MG is more common in females.The etiology of has not yet been determined,but many studies suggest that the pathogenesis of MG is related to heredity,infection,autoantibodies,cytokines and thymic diseases.Clinically,the methods for MG include cholinesterase inhibitors,immunosuppressants,plasma exchange,immunoglobulin and surgical treatment,but the treating effects are not satisfactory.Most of the studies used to focus on the suppression of immune reaction and neuromuscular junction,and lack of researches on the mechanism of myocyte injury caused by abnormal energy metabolism in mitochondria.From the perspective of clinical research and animal experimental research,this article provide scientific basis for the therapy of MG by strengthening the spleen and replenishing qi.In clinical study,by comparing the difference of mRNA expression level of AMP-activated protein kinase(AMPK),p38 mitogen-activated protein kinase(p38MAPK)and cytochrome c oxidase subunitⅣ(COX Ⅳ)in peripheral blood mononuclear cells(PBMC)between MG patients and normal human cells,we explored the relationship between abnormal energy metabolism and myastheina gravis,and further investigate its pathogenesis.In animal experimental research,by replicating the experimental autoimmune myasthenia gravis(EAMG)rat model,watching the ultrastructural changes of skeletal muscle and mitochondria,and detecting the expression of AMPK,p38MAPK and COXⅣ in rat skeletal muscle,we observe the effect of Qiangji Jianli Formula on energy metabolism in EAMG rats and clarify the mechanism of Invigorating spleen and replenishing qi method in improving energy metabolism of skeletal muscle in EAMG rats even further.Methods:1 Clinical StudyAll specimens of patients were taken from August 2018 to February 2019 Spleen and Stomach Disease Department of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine,which were diagnosed by myastheina gravis.A total of 50 MG patients were collected as model group and 50 healthy volunteers were recruited as the normal group.The peripheral blood was extracted from each subject in the morning and the mononuclear cells were separated.The application RT-PCR detection of mRNA expression level of the AMPK,p38MAPK and COXIV in in peripheral blood mononuclear cells and comparison the difference between the two groups.2 Animal experiment2.1 Modeling:72 six-week-old SPF female Lewis rats were selected as experimental subjects.Ten rats were randomly divided into normal control group for normally bred,another 62 rats were selected as model group for subcutaneous injections at different sites of synthetic 97-116 peptide segments of rat-derived acetylcholine alpha subunit for six weeks.After the model being made,8 of them died of overweight symptoms,another 54 patients detected by electromyography and 43 had successfully modeling.2.2 Drug intervention:8 normal rats and 32 EAMG rats were randomly selected by random number table method.Among them,32 EAMG rats were divided into model group and high dose,medium dose,low dose group of Qiangji Jianli Formula(8 rats/group).After grouping,the normal group and the model group were given the equivalent amount of saline.The dosage of Qiangji Jianli Formula in the high,middle and low dose groups was 23.4 g/kg.d,15.6 g/kg.d and 7.8 g/kg.d,respectively.Rats above were treated by intragastric administration with 1 mL/100g for four weeks and its concentration and equivalent dose were converted into body weight.2.3 Measurement of experimental indexes:after the experiment,the weight,general condition and Lennon score of rats were observed and recorded.The rats were sacrificed after 4 weeks of gastric administration and 12 hours of fasting,and the gastrocnemius muscle tissue of the rats was cut off and stored in the refrigerator at-80 C for examination.All the subjects were fasted for 12 hours,then the rat gastrocnemius muscle tissue were shearing and keeping them in-80℃ refrigerator waiting for inspection.Observation the change of Ultrastructure of skeletal muscle and mitochondria under electron microscope,detection the differential expression of mRNA of AMPK,p38MAPK and COX Ⅳ in rat skeletal muscle by RT-PCR,and detection the differential expression of protein of AMPK,p38MAPK and COX Ⅳ in rat skeletal muscle through Western Blot,then comparison the difference between groups.Results:1 Clinical research1.1 General data:50 MG patients and 50 healthy volunteers were collected in this study.There were 19 males and 31 females in MG group,with an average age of(42.58+13.76)years.In addition,there were 25 males and 25 females in normal group,with an average age of(38.92+14.76)years.No statistical difference of age and gender was observed between MG patients and normal persons(P>0.05).1.2 Experimental results:Comparing with normal controls,the mRNA expressions of AMPK,p38MAPK and COXⅣ in peripheral blood mononuclear cells of MG patients were significantly lower,with a statistically significant difference(P<0.05).2 Animal experimental study2.1 General condition:The EAMG rats withered hair and weight decreased by modeling.After treatment with Qiangji Jianli Formula,the hair color was improved and the body weight increased.2.2 Electron microscopic observation of skeletal muscle and mitochondria in rats:It has been found that EAMG rats had skeletal muscle fibers disorders,swelling,vacuolation and cristae rupture of subsarcolemmal mitochondria,and the abnormal morphology of skeletal muscle and mitochondria was improved after strong muscle strengthening therapy.2.3 The Changes of the mRNA expression for AMPK,p38MAPK and COXⅣ in skeletal muscle of rats in groups:The experimental results showed that the expression of COXⅣ in model group was lower than that in normal group(P<0.05),but the expression of AMPK and p38MAPK in model group was not significantly different from that in normal group(P>0.05).After oral administration of Qiangji Jianli Decoction,the expression level of COX Ⅳ in high dose rats was higher than that in model group(P<0.05).The expression of COX Ⅳ in middle dose group and low dose group was higher than that in model group,but there was no significant difference(P>0.05).There was no significant difference in the expression of AMPK and p38MAPK mRNA between each dose group and model group(P>0.05).2.4 The expression of AMPK,p-AMPK,p38MAPK,p-p38MAPK and COX Ⅳ protein in skeletal muscle of rats in each group:The results showed that the protein expression of p-AMPK,p-p38MAPK and COX Ⅳ in the model group was significantly lower than that in the normal group(P<0.05).The expression of p-AMPK,p-p38MAPK and COX Ⅳ protein in skeletal muscle of high dose Qiangji Jianlifang rats was higher than that of model group(P<0.05),and the expression of p-p38MAPK and COX Ⅳ protein in skeletal muscle of middle dose group was higher than that of model group(P<0.05),The expression of p-AMPK protein in skeletal muscle of rats in middle dose group was not significantly different from that in model group(P>0.05).The expression of p-AMPK,p-p38MAPK and COX Ⅳ protein in low dose group was not significantly different from that in model group(P>0.05).Conclus ion:1.Clinical researchThe mRNA expression levels of AMPK,p38MAPK and COXⅣ in peripheral blood of patients with myasthenia gravis were lower than those in healthy volunteers,wihch indicating that abnormal mRNA expression of AMPK,p38MAPK and COXⅣ might play a role in the pathogenesis of myasthenia gravis.2.Animal experimental research2.1 EAMG rat model can be successfully reproduced by subcutaneous injections at different sites of synthetic 97-116 peptide segments of rat-derived acetylcholine alpha2.2 Qiangji Jianli Formula can improve the withered yellow hair color,muscle weakness,weight loss and other symptoms of EAMG rats.2.3 The mRNA expression of COXⅣand the protein expressions of p-AMPK,p-p38MAPK and COXⅣ were decreased in EAMG rats.However,after treatment with Qiangji Jianli Formula,the expression culd increased.Therefore,we speculated that the pathogenesis of myasthenia gravis may be related to AMPK,p38MAPK,and COXⅣ,besides,the Qiangji Jianli Formula may play a role by regulating the related pathways in which they participate. |
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