| Objective: on the basis of investigating the effect of Jianpi Yiqi Busui prescription on the mechanism of mitochondrial fusion and division related regulatory proteins mfn1 / 2,OPA1,drp1 and FIS1 in experimental autoimmune myasthenia gravis rats,and testing the acetylcholinesterase inhibitory activity of Jianpi Yiqi Busui prescription and its flavor in terms of the symptomatic treatment of myasthenia gravis,this paper explains the mechanism of Jianpi Yiqi Busui method in the treatment of myasthenia gravis,and evaluates the internal quality of Jian Pi Yiqi Busui prescription,so as to provide scientific basis for ensuring stable clinical efficacy;Methods: 1.The experimental autoimmune myasthenia gravis rat model(EAMG)was established by injecting immune antigen.The effect of Jian Pi Yiqi Busui prescriptionon the experimental autoimmune myasthenia gravis animal model was evaluated by observing the clinical symptoms,weight changes and detecting the level of acetylcholine receptor antibody(ACh R AB)in the peripheral blood of the model rats.2.The gastrocnemius muscle tissue samples of rats in high-dose group,medium dose group,low-dose group,positive drug group(prednisone group),model group and other groups of Jianpi Yiqi Busui prescription were collected.The samples were fixed,dehydrated,paraffin embedded and sectioned,dewaxed and hydrated,antigen repair,sample fixation,serum blocking,first antibody and second antibody incubation,DAB staining,re-staining,and sealing,and the flow was performed according to the immunohistochemical method The average optical density of protein expression was measured by microscope observation and image analysis system.The expression differences of mfn1,Mfn2,OPA1,drp1 and FIS1 were statistically analyzed.3.The spleen,thymus and gastrocnemius muscle of the rats in the above experimental groups were taken and examined by HE staining and image analysis system to analyze the histology and Pathomorphology,and the cross-sectional area of gastrocnemius muscle fiber was measured.4.Ellman method was used to test and study the inhibitory activity of Jianpi Yiqi Busui prescription and its traditional Chinese medicine on theactivity of acetylcholinesterase,and the correlation between effective components and pharmacodynamic activity was also studied.5.HPLC-ELSD fingerprint analysis and multicomponent quantitative analysis were used to evaluate the quality of the drug.Results: 1.Model establishment: after the second booster immunization 45 days after the first immunization,the symptoms of EAMG model rats were further aggravated,such as shortness of breath,more severe shaking,unable to lift the neck,head close to the table,touching the maxilla.Compared with the normal control group,the weight of EAMG group was further reduced,and the secretion level of ACh R AB in peripheral serum of EAMG group was significantly increased(p<0.0001)。2.Weight and symptoms of EAMG: compared with EAMG group,the weight of rats in high,medium and low dose groups of traditional Chinese medicine increased from the 20 th day of administration,which was statistically different from that in EAMG group(P < 0.01).It sindicated that Jianpi Yiqi Busui prescription could improve the weight and clinical symptoms of EAMG.3.The level of ACh R Aβ antibody in peripheral serum of EAMG group: the level of ACh R AB antibody in peripheral serum of EAMG group was always higher than that of control group during the whole period of intragastric administration.Compared with EAMG,the levels of ACh R AB in peripheral serum of rats in each dose group of TCM were significantly decreased,and the levels of ACh R AB in peripheral serum of rats in high-dose group of TCM were similar to those in prednisone group,there was no significant difference(P = 0.22 > 0.05).(P = 0.22 >0.05).4.The experimental results of regulating the expression level of mitochondrial fusion and division proteins: the results of immunohistochemical study on the expression level of the five proteins mfn1,Mfn2,OPA1,drp1,FIS1 which make mitochondria in a stable state showed that the labeled positive protein was brown or light yellow,and the negative protein was not yellow.In the control group,the expression of mitochondrial dynamics related proteins was higher,the positive degree was higher,and the distribution area was larger.Compared with the control group,the expression of five related proteins in the model group decreased(P < 0.01);after administration of Jianpi Yiqi Busui prescription,the expression of five related proteins in mitochondria increased,making the mitochondrial fusion and division return to dynamic balance,maintaining the stability of mitochondrial morphology and biological synthesis,ensuring the stable play of mitochondria,and promoting the recovery of mitochondrial functional mechanism.5.Effect of Jianpi Yiqi Busui prescription on histopathological morphology of immune organs results: in normal spleen group,spleen contains a large number of lymphoid tissue,and spleen parenchyma is divided into three parts: medulla,white medulla and marginal area;white medulla is mainly composed of lymphoid tissue with dense lymphocytes,which can be divided into two parts:periarterial lymph sheath and lymph nodes.The periarterial lymphatic sheath consists of a large number of T cells and a small number of macrophages and crisscross cells.The lymph nodes were composed of a large number of B cells;in the blank control group,the spleen structure was normal,and there was no obvious germinal center.In the model group,the spleen was atrophic,the content of lymphocytes in the spleen was significantly reduced,the lymphatic sheath and lymph nodes around the white intramedullary artery were residual or almost disappeared,the cell composition in the red pulp was significantly reduced,a large amount of hemosiderin was distributed,and the blood storage and hematopoietic function of the spleen were seriously damaged.The area of lymph sheath and lymph node around the white pulp artery in Jianpi Yiqi Busui prescription and positive drug groups and spleen increased in varying degrees.The white pulp component increased more in high and middle dose group,and the white pulp component increased in positive drug and low dose group.The overall red pulp function improved,and the blood storage and hematopoietic function increased.Thymus belongs to the central lymphoid organ and is the site of early differentiation of lymphocytes.Lymphoid stem cells divide and differentiate in the cells,and can continuously transport virgin lymphocytes to the surrounding lymphoid organs and lymphoid tissues.A thin layer of connective tissue on the surface of the thymus extends into the thymus parenchyma to form interlobular septum,which separates the thymus and forms thymic lobules.Each lobule is divided into cortex and medulla.Thymocytes in the cortex are dense and stained deeply.There are more epithelial cells in the medulla and stained lightly.Normal control group: the cortex and medulla of thymus tissue were stained clearly,the cortex was rich in thymocytes,which were the precursors of T cells;the medulla contained more thymic epithelial cells,occasionally thymic corpuscles.In the model group,the thymus was atrophic,the cell composition was significantly reduced,the differentiation of lymphocytes in medulla was impaired,and the apoptotic cell composition was increased,which could form a starry sky.The cortical components decreased and became thinner.Provide thymus dysfunction.In the positive drug group and the administration group,the starry sky phenomenon of cell apoptosis in the thymus medulla was significantly reduced,the components of cortex and medulla cells were increased,and the thymus function was increased.The effect of high and middle dose of drugs on increasing the components of thymus cells was better,and the effect of positive drug group was similar to that of low dose group.Gastrocnemius muscle tissue: in the normal group,the muscle section was red stained,and obvious muscle fibers could be seen in the muscle cells.The muscle membrane was relativelycomplete and clear,and the stripes on the muscle fibers were bright and dark,and the bright and dark bands and Z-lines could be seen.In the model group,the atrophic muscle fibers shrank,the color was lighter,the edge of muscle fibers became straight or depressed,and the gap between muscle fibers was widened.In addition,the number,shape and location of sarcolemma nuclei were also abnormal.In the transverse section,the number of sarcolemma nuclei around the muscle fibers increased,while in the longitudinal section,the sarcolemma nuclei were dense or arranged in chains,and nuclear translocation could be seen.In cross section,the nucleus of sarcolemma is located in the central part of the muscle fiber,which is called the nuclear shift.Light staining of muscle cells,general atrophy,fibrous degeneration and edema,intermuscular looseness,light and dark bands and Z-line loose and blurred disappeared,cross-sectional area of muscle fibers reduced,and the number of cells and muscle cells increased in a single visual field.In the positive drug group and the administration group,the integrity,clarity and texture uniformity of muscle membrane showed different degrees of recovery,the degree of muscle staining was enhanced,and the clarity of bright band,dark band and Z-line structure was enhanced,which improved muscle functional metabolism and increased the recovery of muscle cell morphology and function.The most complete and clear muscle membrane was recovered,the muscle cells were more orderly,the fibrous degeneration and edema were relieved.6.Study and results on the inhibitory activity of acetylcholinesterase: the extract of Jianpi Yiqi Busui prescription and its active ingredients have a good inhibitory effect on acetylcholinesterase.7.Study on internal quality evaluation of Jianpi Yiqi Busui prescription: HPLC fingerprint has good similarity,13 compounds in 24 fingerprint peaks were identified by LC-MS,and the content determination method is accurate and reliable.The results of cluster analysis and principal component analysis showed that hesperidin,Acteoside and Lobetyolin were important chemical markers.Conclusion: the results of this study show that this prescription can regulate the expression of five proteins related to mitochondrial fusion and fission,restore the dynamic balance of mitochondrial fusion and division,maintain the stability of mitochondrial morphology and biosynthesis,ensure the stable play of mitochondria,restore the function of mitochondrial energy factory,and ensure the normal life movement of cells.In this study,through the study on the influence of mitochondrial fusion and division on energy metabolism,in addition to immunology,we explained the multi-level and multi-target mechanism of Jianpi Yiqi Busui prescription in the treatment of myasthenia gravis from another perspective.The results of histopathology showed that: in the positive drug group and the administration group,the immune cells in the spleen and the function of blood storage and hematopoiesis were increased;the cell components in the thymus(the precursor of T cells)were increased;this indicated that the immunomodulatory function of the prescription had achieved substantial pathological improvement;the metabolism of muscle function was improved,and the recovery of muscle cell morphology and function was increased.So as to play a therapeutic effect on myasthenia gravis.MG is an autoimmune disease,which is mainly caused by the blocking of acetylcholine receptor binding site in postsynaptic nerve muscle by auto circulating antibody.Acetylcholinesterase inhibitor is a symptomatic treatment,which slows down the hydrolysis of acetylcholine,makes more acetylcholine activate normal acetylcholine receptor,and makes patients’ symptoms get short-term relief and improvement.So far,acetylcholinesterase inhibitors are the first-line and the first step in the treatment of Mg.For example,pyristigmine has been safely used in clinical practice for more than 60 years,but it has great toxic and side effects,and natural products have high safety against acetylcholinesterase.Based on this,this project carried out the research on the in vitro pharmacodynamic activity of Jianpi Yiqi Busui prescription extract,Chinese herbal medicine and monomer compounds in the symptomatic treatment.The results showed that this prescription has a good inhibition of acetylcholinesterase activity,which is also one of the multi-target mechanism of this prescription in the treatment of MG. |
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