The Characteristic Of TCM Synderome Of Wilson Disease Combined With Abnormal Bone Metabolism And Clinical Experimental Study Of Invigorating The Kidney Eliminating Phlegm Resolving Stasis Recipetreatment

1 Objective1.1 Combination with clinical observation,the distribution characteristics of TCM syndromes in patients with Wilson's disease accompanied by abnormal bone metabolism were explored by using the combination of four traditional Chinese medicine diagnosis and treatment based on syndrome differentiation.1.2 Clinical intervention of tonifying kidney,removing phlegm and removing blood stasis prescription was used to understand the clinical therapeutic effect of traditional Chinese medicine on patients with Wilson's disease accompanied by abnormal bone metabolism.1.3 Through the animal experiment observed the pathological morphological changes,TX mouse model femoral organization and ascertain the kidney and phlegm and eliminating stasis party of TX mice bone metabolic effects change the mechanism of action,whether study of kidney and phlegm and eliminating stasis TX mouse bone tissue growth has certain protection mechanism,so as to intervention of Chinese medicine in the treatment of Wilson disease combined bone metabolic abnormalities of patients to provide reliable theoretical basis for experiment.2 Methods2.1 Syndrome investigationFrom January 2019 to December 2019,138 cases of Wilson's disease in the Anhui hospital of traditional Chinese medicine were screened and collected from the Wilson's disease group?WD group?and 102 cases of Wilson's disease?BD group?with abnormal bone metabolism after completing relevant examinations in the order of visit.For patients meeting the inclusion criteria,TCM symptoms and signs were recorded and uniformly included in the EXCEL spreadsheet.2.2 Clinical research Sixty patients with hepatolenticular degeneration with abnormal bone metabolism from January 2019 to December 2019,who were admitted to the department of encephalopathy,the randomized method was divided into the treatment group and the control group,with an average of 30 patients in each group in Anhui provincial hospital of traditional Chinese medicine.Among them,the treatment plan of the control group was the routine western medicine to protect liver and remove copper,and the specific treatment plan is to add or subtract prescription of traditional Chinese medicine with the function of tonifying kidney,removing phlegm and removing blood stasis on the basis of the treatment of liver and remove copper.On this basis,the changes of clinical syndrome scores and the clinical improvement of abnormal symptoms of bone metabolism in the control group and the treatment group were observed respectively.2.3 Genotype detectionFrom 7 to 10 days of age of SPF C3He ATP7bt^x-jmice toes extracted genome,purification of samples from the organization,to obtain pure DNA,according to the samples of primers for PCR amplification,amplification products shall be carried out in accordance with the experimental steps and degeneration,modified by annealing,extension,repair,and extended,after cutting rubber cutting gel purification,validated by Sanger sequencing.2.4 Experimental researchUnder the SPF level laboratory environment,50 c3he-atp7bt^x-jmice were adaptively fed for 14 days,and in a random way divide the mice into blank group,model group,DMSA group,kidney-tonifying and dispelling phlegm and removing blood stasis group,kidney-tonifying and removing phlegm and removing stasis group,and DMSA group,totaling 5 groups,with an average of 10 mice per group.During the experiment,the daily dose of the drug was converted according to the body surface area,which was about 1:9.Specifically,mice in the drug group were given gavage treatment with the drug dose of 20ml liquid/kg/day every morning time,and given gavage treatment with the same volume of normal saline once a day for the blank group and Wilson disease model group,and lasts for 14 days.In lavage period after the end of the first day of,be10%chloral hydrate anesthesia in mice,cervical dislocation was carried out to put to death in mice,and take a side,with a cut skin,take its bilateral femoral organization,soft tissue,eliminate the femoral surface in formaldehyde solution containing 4%neutral precooling?4??of the EP tube fixed more than 48 h,decalcified in 10%EDTA solution,once every 3 days in decalcified fluid,use the big beard needle test tissue for the hard and soft,with needle can be easily through the decalcified adequately.Before removing calcium treatment soaking specimens for appropriate water and running water washing,in order to reduce the formaldehyde residue epicuticular subsequent production effect of the intervention,after Dai Zhi paraffin section,standby HE staining to observe the histopathological changes of shape and structure of the femur in mice,and using his red staining method further analyze stripped of cartilage tissue in mice and growth;The expression levels of Osteocalcin?BPG?and Osteoprotegerin?OPG?in mouse femur were detected by immunohistochemistry.3 Results3.1 Studies on TCM syndrome distribution rate,score,composite syndrome and risk coefficient of the tested patients3.1.1 Results of distribution of clinical TCM syndrome elementsThe distribution rate of blood stasis,phlegm and dampness,kidney Yin deficiency and kidney Yang deficiency syndrome in BD group was more higher than that in WD group?P<0.05?.The factor distribution rate of syndromes of liver Yin deficiency,heat,spleen Yang deficiency and blood deficiency in WD group was more higher than that in BD group?P<0.01 or P<0.05?.There was no obvious difference in the distribution of qi stagnation and qi deficiency syndrome?P>0.05?.3.1.2 Comparison of TCM syndrome scores between the two groups Compared with WD group,the clinical TCM syndrome scores of patients with blood stasis,phlegm and dampness,kidney Yin deficiency and kidney Yang deficiency in BD group were significantly higher?P<0.01?.Compared with BD group,the clinical TCM syndrome scores of heat,spleen Yang deficiency and liver Yin deficiency in WD group were higher?P<0.01 or P<0.05?.There was no statistically significant difference in the scores of clinical TCM syndromes of qi deficiency,qi stagnation,Yang hyperactivity and blood deficiency?P>0.05?.3.1.3 The distribution of complex syndrome types was includedIn the distribution of multiple syndromes,the proportion of BD group was higher than that of WD group?P<0.01?.On the distribution of single syndrome,the proportion of WD group was higher than that of BD group?P<0.01?.However,in terms of the two syndrome distributions,there was no statistically significant difference in the proportion of WD group and BD group?P>0.05?.3.1.4 Study on the risk coefficient of Wilson's disease with abnormal bone metabolism by TCM syndromeLogistic regression analysis was used to study the risk factors of various TCM syndromes for hepatolandular degeneration with abnormal bone metabolism.It can be seen that the regression coefficients of blood stasis,phlegm and dampness,kidney Yang deficiency and kidney Yin deficiency were 2.609,3.242,2.801 and 3.121,respectively,and all had statistical significance?P<0.01 or P<0.05?.3.2 Changes of TCM syndrome score before and after treatmentBefore treatment,there was no significant difference in clinical TCM syndrome score between the two groups?P>0.05?.After treatment,the clinical TCM syndrome score of the two groups decreased significantly,and the score score of the treatment group decreased more obvious,with statistically significant differences?P<0.01?.3.3 Results of gene testing in miceSanger sequencing method was used to detect the genotype of the newborn mouse samples.The results showed that the wild-type mice showed single peak sequencing peak map and no mutant base G.The heterozygous mice were bimodal sequencing peaks.Peak map of mutant base A sequencing in homozygous mice.3.4 Conclusions of experimental research3.4.1 The effect of tonifying kidney removing phlegm and removing blood stasis prescription on pathological changes of femur in TX miceHE staining was used to observe the pathological structure of the femur tissue of TX mice.It can be seen that the femur tissue structure of the blank group was basically complete and orderly,and the bone trabecular structure was compact and evenly distributed.Compared with the blank group,the femur tissue structure of mice in the Wilson disease model group was changed and the arrangement was disordered,the trabecular bone was significantly sparse and loose,the number was reduced,the break point was more,the bone marrow cavity was enlarged,and even the bone marrow region without bone trabecular appeared.However,in the DMSA group,kidney tonifying and removing phlegm and removing stasis group,and DMSA+kidney tonifying and removing phlegm and removing stasis group,the degree of pathological disorder of the femur was less than that of the Wilson disease model group,and the pathological changes of the DMSA+kidney tonifying and removing phlegm and removing stasis group were less than that of the DMSA group and kidney tonifying and removing phlegm and removing stasis group.The changes of cartilage pathological structure in the femur tissue of TX mice were further observed by saffron staining:the cartilage tissue in the blank group had smooth surface,uniform width,no cracks or erosion in the whole cartilage layer,complete connectivity,intact chondrocytes,clear boundaries,normal nuclei,no atrophy,and good development.Compared with the blank group,the mice in the Wilson disease model group had cartilage tissue destruction and fracture,with thinning thickness,erosion,uneven and rough surface layer,loss of surface cartilage,and disordered cell arrangement,showing obvious signs of tissue structure destruction.However,the cartilage tissue of mice in DMSA group,kidneytonifying and removing phlegm and removing stasis group and DMSA+kidneytonifying and removing phlegm and removing stasis group had good development,smooth and complete cartilage surface,and the cartilage morphology of TX mice in DMSA+kidneytonifying and removing phlegm and removing stasis group was better than that in DMSA group and kidneytonifying and removing phlegm and removing stasis group.3.4.2 Effects of tonifying kidney,removing phlegm and removing blood stasis prescription on the expression of BPG protein and OPG protein and the level of average optical density in the femur tissue of TX miceCombined protein results:compared with blank group,the femur in Wilson's disease model mice tissues combined protein expression level decreased obviously,reduce the average optical density?P<0.01?,compared with Wilson's disease model group,group of DMSA,kidney and phlegm and remove blood stasis and DMSA+kidney and phlegm and eliminating stasis more combined protein expression level of the party group,the average optical density increased?P<0.01?,group compared with DMSA+kidney and phlegm and remove blood stasis,DMSA groups with kidney and phlegm and remove blood stasis combined protein expression level decreased,to reduce the average optical density?P<0.05?.OPG protein results:Compared with the blank group,the femur in Wilson's disease model mice in the organization of OPG protein expression level decreased obviously,reduce the average optical density?P<0.01?,compared with model group Wilson disease,DMSA,kidney and phlegm and remove blood stasis group and DMSA+kidney and phlegm and remove blood stasis group of OPG protein expression level increased,the average optical density increased?P<0.01?,group compared with DMSA+kidney and phlegm and remove blood stasis,DMSA with kidney and phlegm and remove blood stasis group of OPG protein expression level decreased,The average optical density decreased?P<0.05?,and the expression level of OPG protein in DMSA group and the group of tonifying kidney,removing phlegm and removing blood stasis was similar,with no significant difference.4.Conclusion4.1 Blood stasis,phlegm and dampness,kidney Yin deficiency and kidney Yang deficiency are the main TCM syndromes in patients with Wilson's disease with abnormal bone metabolism.4.2 Blood stasis,phlegm and dampness,kidney Yin deficiency and kidney Yang deficiency are the main risk factors for abnormal bone metabolism in Wilson's disease.4.3 Wilson's disease is accompanied by gradual aggravation of bone injury,and the syndrome of hepatolenticular degeneration with abnormal bone metabolism becomes more complex.4.4 Tonifying kidney,removing phlegm and removing blood stasis can effectively improve the TCM syndrome score of Wilson's disease patients with abnormal bone metabolism and alleviate the severity of their clinical symptoms.4.5 In the genetic test of mice,there was no mutation of the unimodal map base G in the wild type mice,while the unimodal map base G in the heterozygous type mice and the unimodal map base G in the pure type mice was mutated to base A.4.6 TX mice had a certain degree of damage to the structure and morphology of the femur tissue and bone cell development,which affected the balance of bone metabolism.4.7 Tonifying kidney,removing phlegm and removing blood stasis can improve the expression levels of BPG protein and OPG protein in TX mice bone tissue to a certain extent,and improve the bone metabolic disorder of TX mice.