Preliminary Discussion On The Relationship Between The Tuberculosis Bacili InhA Mutation And Molecular Mechanisms Of Resisting Isoniazid Pulmonary Tuberculosis By Fei Langkang

Objective:Researching on the molecular mechanisms of using traditional Chinese medicine compound Feilaokang to treat the mouses which can resist isoniazid pulmonary tuberculosis, from the aspect of molecular biology, exploring the relationship between the therapeutic effect of it and inhA genetic mutation, and offering some research ideas on using Chinese traditional medicine to treat drug-resistant pulmonary tuberculosis.Methods:The study take 80 mouses first,through caudal vein injection to build the model of isoniazid-resistant pulmonary tuberculosis animals, diving them into 4 groups randomly:model group,isoniazid group, feilaokang group and isoniazid combined Feilangkang group.Treating them with physiological saline, isoniazid, Feilaokang,isoniazid combined feilangkang lavage respectively for 56 days;Then take 20 mouses,through caudal vein to inject H37RV standard mycobacterium tuberculosis bacterial suspension, build the model of H37RV standard bacteria mouses,use physiological saline to lavage them for 56 days; Then take 20 mouses,through caudal vein to inject physiological saline build the model of blank mouses,use physiological saline to lavage them for 56 days; Comparing the each group’s general pathological signs and the weight changes before and after the treatment, after 56-day moulding, take out the lung tissue to do the bacterial culture; After treatment, observing the pathological changes of mouses’ lungs tissue under naked eye and under the microscope.Through whole genome sequencing technology to sequence the mycobacterium tuberculosis genome DNA in posttreatment mouses lung tissue, regarding the standard strain (H37RV) as reference Sequence,then screening,comparing,analysing them.Adopting SPSS21.0 statistical software to analyse the measuring and counting data in the experiment.Adopting the softwares such as bwa(version 0.7.8、samtools、Picard and so on to deal with the data and compare the expression results of bases and genome.Result:1.Before moulding,the weights of mouses of blank group, isoniazid-resistant strain group, standard strain group had no obvious difference (P> 0.05),and each mouse’s weight presented normal distribution;After moulding,the mouses of blank group, isoniazid-resistant group and standard strain group gained some weight,comparing the weights of mouses of blank group,isoniazid-resistant strain group, standard strain group,the difference of weights has statistical significance (P<0.05),isoniazid-resistant strain group and standard strain group gained less weight than the blank group.2. Before and after the treatments,through the comparisons of weight changes of the mouse,there is no statistical significance in each group’s mouses weights before the treatment (P>0.05),there is statistical significance in each group’s mouses weights after the treatment (P<0.05),the difference between standard strain group and model group has no statistical significance (P>0.05),the difference among isoniazid group, Feilaokang group, isoniazid combined feilangkang group and model group has statistical significance (P<0.05);Observing the shapes of mouses’lungs through naked eye,there were obvious changes in the shapes of standard strain group and model group,all the mouses presented tubercles,and there were only a part of mouses in isoniazid group, Feilaokang group, isoniazid combined feilangkang group presented tubercles. Contrasting the number of tubercles of each group mouses, standard strain group and model group has no statistical significance (P>0.05); Isoniazid group and model group has statistical significance (P<0.05); Feilaokang group and and model group has statistical significance (P<0.05); Isoniazid combined Feilangkang group and model group has statistical significance (P<0.05); Feilaokang group, model group and isoniazid combined Feilangkang group has no statistical significance (P<0.05); Under microscope,compared with model group, treatment group lungs inflammation relieved obviously; The study found that there are possible 3 mutation base locus related to inhA genetic mutation, located in 1673425th、1676290th、1678706th of genome respectively,and the corresponding bases are C、C、A, then mutate into T、 A、C. The location s of bases mutation are upstream、downstream、downstream of coding sequence respectively,and this mutation is located in non-coding regions,the changes resulted in the coding sequence of genome are at 777th、1279th、3695th respectively. Contrasting the locus of each group base,the locations of 3 bases in standard strain and the reference bases are the same,the isoniazid-resistant tuberculosis bacili of model group, isoniazid group, feilaokang group, isoniazid combined Feilangkang group and inhA base had mutations, and the locations of mutation、the coding sequence of genome and genes are same.Conclusion:1.Treating isoniazid-resistant pulmonary tuberculosis model mouses with feilaokang can improve the general pathological signs and tissues’pathogenic changes of mouses significantly, relieve the lung inflammations reactions, and offer more basis of using clinical application to treat drug-resistant pulmonary tuberculosis.2.The location s of bases mutation are upstream、downstream、 downstream of coding sequence respectively,and this mutation is located in non-coding regions,the changes resulted in the coding sequence of genome are at 777th、1279th、3695th respectively.3.The relationship between the molecular mechanisms of resisting isoniazid pulmonary tuberculosis by Feilaokang and tuberculosis bacili inhA genetic mutation are not strong,this provides reference materials for the future study of using Traditional Chinese medicine compound to resist tubercles.