Clinical And Experimental Study On The Mechanism Of Yanghe Decoction In The Treatment Of Spinal Tuberculosis Based On The Theory Of Strengthening Body Resistance And Eliminate Pathogenic Factors In TCM

BackgroundBone tuberculosis is one of the most common extrapulmonary tuberculosis,and spinal tuberculosis accounts for more than 50%of bone and joint tuberculosis.Improper treatment will lead to severe kyphosis and neurological dysfunction,and even paraplegia,which is extremely harmful to human health.Spinal tuberculosis belongs to the category of"gangrene"and"phlegm"in Traditional Chinese medicine.Yanghe decoction is a classic prescription for the treatment of gangrene and phlegm.It is widely used in the treatment of orthopedic diseases,especially in the treatment of bone tuberculosis,with remarkable curative effect.However,the mechanism of its action in the treatment of bone tuberculosis is still lacking.This topic intends to explore the immunological mechanism of Yanghe Decoction in the treatment of spinal tuberculosis from two aspects of clinical research and animal experiments,based on the theory of TCM"strengthening body resistance and eliminate pathogenic factors",so as to provide new ideas for the development and use of TCM to prevent and treat such diseases.Part I:Clinical research(1)Research on the clinical characteristics and immune level differences of different types of spinal tuberculosisObjective:To observe the clinical characteristics and immune levels of patients with yang-deficiency type and yin-deficiency type spinal tuberculosis,compare the differences between the two groups,and provide an objective basis for TCM clinical dialectical treatment.Methods:A cross-sectional clinical research method was used to collect clinical data from patients with spinal tuberculosis,including demographic information,clinical symptoms,imaging data,routine laboratory examinations and T cell subset detection.According to the TCM dialectical classification,the patients finally included in the study were divided into two groups:yang deficiency type and yin deficiency type,and the differences of the above indicators between the two groups were compared.Results:A total of 113 patients were finally included in this study,including 66 patients(58.4%)in the Yang deficiency group and 42(41.6%)in the Yin deficiency group;there was no significant difference in gender distribution and age between the two groups in terms of demographic characteristics;The average course of disease was 5.67±2.16 months,which was higher than that of the Yin-deficiency group,which was 4.17±2.66months;the BMI index of the Yang-deficiency group was 16.73±3.87(kg/m~2),which was lower than that of the Yin-deficiency group,which was19.12±5.05(kg/m~2);symptom comparison:VAS score between the two groups,ODI index and Frankel grade of spinal cord injury were not significantly different;imaging comparison:there was no significant difference in the lesion location and the number of involved vertebral bodies between the two groups,but the incidence of abscess in the Yang-deficiency group was higher than that in the Yin-deficiency group;Comparison of laboratory examinations:The incidences of anemia and low albumin in the yang deficiency group were higher than those in the yin deficiency group.The overall level of erythrocyte sedimentation rate in the Yang deficiency group was lower than that in the Yin deficiency group(p<0.05);comparison of T cell subsets:CD4+/CD8+in the Yang deficiency group was lower than that in the Yin deficiency group,and the difference was statistically significant.Conclusion:There are certain differences between the yang-deficiency type and yin-deficiency type of spinal tuberculosis.Patients with yang deficiency have a longer course of disease,a higher incidence of paravertebral abscess,and a lower nutritional status and immune level,which can provide a certain reference for clinical dialectics.(2)Clinical efficacy of Yanghe decoction assisted by surgery in the treatment of spinal tuberculosis and its effect on immunityObjective:To observe the clinical efficacy of Yanghe decoction in the treatment of spinal tuberculosis,and to compare the differences in T cell subsets and Th1/Th2 cytokine expression between the two groups before and after treatment.Methods:66 patients with yang-deficiency type spinal tuberculosis were randomly divided into the observation group(Yanghe decoction+surgery+chemotherapy)and the control group(surgery+chemotherapy),33people in each group,both groups were treated for 12 weeks,before treatment(T0),The patients were clinically evaluated at 5 time points one week after operation(T1),4 weeks after operation(T2),8 weeks after operation(T3),and 12 weeks after operation(T4),and peripheral blood was collected for detection and analysis,and the two groups were compared.Baseline data,pain and disability scores,erythrocyte sedimentation rate,proportion of T cell subsets,TCM syndrome scores,and TCM efficacy.The adverse reactions of the two groups during treatment were observed.Results:In the observation group,2 cases dropped out and 1 case dropped out.In the control group,1 case dropped out,2 cases dropped out,and each group completed 30 cases;there was no significant difference in the baseline data between the two groups,and they were comparable.Comparison of VAS scores:The VAS scores of the two groups at T0,T1,T2,T3,and T4 all showed a downward trend over time.At T2 and T3,the observation group was lower than the control group;ODI score comparison:The ODI scores of the two groups changed over time All showed a downward trend.At T2,T3,and T4,the observation group was lower than the control group;ESR comparison:The ESR levels of the two groups showed a downward trend with time.At T2,the ESR index of the observation group was lower than that of the control group.Comparison of TCM syndrome scores:The TCM syndrome scores of the two groups increased at T1,and then gradually decreased.At T2 and T3,the observation group was lower than the control group.Comparison of T lymphocyte subsets:CD4+,CD4+/CD8+in the observation group at T4 Compared with before,the levels of CD4+and CD4+/CD8+in the observation group were higher than those in the control group.Th1/Th2 cytokine comparison:the level of IL-10 in the observation group at T4 was lower than that at T0,and lower than that in the control group;the level of IFN-?was higher than that at T0,and higher than that in the control group.Comparison of traditional Chinese medicine curative effect:T4 observation group had a marked rate of 43.3%higher than that of control group,20.0%;in terms of safety,there were no serious adverse reactions in the two groups,and the incidence of adverse reactions in the observation group was lower than that in the control group.Conclusion:Yanghe decoction assisted by surgery and chemotherapy in the treatment of spinal tuberculosis can significantly improve the clinical symptoms of patients,relieve inflammation,improve the immune level of patients,and help restore the balance of Th1/Th2.Part II:Experimental Research(1)Construction of a mouse model of Mycobacterium tuberculosis infection and identification of medicinal components of Yanghe DecoctionObjective:Establish and verify the mouse model of Mycobacterium tuberculosis infection,identify the ingredients and stability of the drug Yanghe Decoction used in the experiment,and lay the foundation for the in vivo experimental study of Yanghe Decoction in the treatment of spinal tuberculosis.Methods:Twelve C57BL/6 mice were randomly divided into 3 groups:blank control group(Sham group),H37Ra strain infection model group(TB group),and positive drug isoniazid treatment group(INH group),with 4 mice in each group.The TB group and the INH group were injected with 0.2 m L of attenuated Mycobacterium tuberculosis H37Ra strain 106CFU through the tail vein;the Sham group was injected with 0.2 m L of normal saline through the tail vein.After 2 weeks of infection,intragastric administration was started.The INH group was given 0.2 ml of isoniazid to prepare the liquid;the Sham group and the TB group were given 0.2 ml of normal saline.After continuous administration for 4 weeks,all mice were sacrificed and operated aseptically The lungs of the mice were dissected,the left lung was tested for the amount of bacteria in the tissue,the right lung was subjected to pathological sectioning and acid-fast staining,and the general conditions of the mice were observed and recorded during the administration period.The active ingredients and stability of Yanghe decoction were identified by high performance liquid chromatography.Results:A total of 12 mice in this group completed the experiment.One mouse in the model group had redness and swelling at the tail puncture site,which gradually healed after 1 week.After 6 weeks of H37Ra infection(4weeks of INH intervention),the bacterial load in the lung tissue of the Sham group,the TB group and the INH group was 0,4.96±0.26,2.56±0.48(Lg CFU/m L),respectively;compared with the TB group,the INH group The amount of bacteria in the lung tissue was significantly reduced;pathological examination showed that no typical tuberculosis lesions were found in the lung tissue of the mice after infection,and inflammatory cells were aggregated in the lung tissue.The degree was significantly reduced compared with the TB group.Acid-fast staining of lung sections in the TB group showed positive bacterial staining;6 independent batches of Yanghe decoction used in this study were analyzed by HPLC.Overlaid chromatograms indicated that the stability of Yanghetang was confirmed.Seven main chemical components were identified by HPLC,and the identified chemical components were oleanolic acid,ephedrine hydrochloride,sinapine thiocyanate,liquiritin,cinnamaldehyde,wogonin and 6-gingerol.Conclusion:The H37Ra strain infection model of mice was successfully constructed,and the response to isoniazid drug treatment was obvious.The active ingredients of Yanghe decoction used in this study are stable and can be used for further experimental research.(2)Inhibition of Mycobacterium tuberculosis and regulation of immune indexes by Yanghe decoction in miceObjective:To observe the effect of Yanghe decoction combined with isoniazid treatment on the tissue organ index,tissue load,and histopathological manifestations of tuberculosis-infected mice,as well as the regulation of spleen T cell factors INF-?and IL-10 in mice.Methods:Forty C57BL/6 mice were randomly divided into 5 groups:blank control group(Sham group),H37Ra strain infection model group(TB group),positive drug treatment group(INH group),Yanghe decoction treatment group(YHT group);Yanghe decoction+isoniazid treatment group(YHT+INH group),8 mice in each group;except the Sham group,mice in the other four groups were injected with 0.2 m L of H37Ra strain 106CFU into the tail vein for infection modeling.The administration started 2 weeks after infection.The INH group was given 0.2 ml of isoniazid solution by gavage,the YHT group was given 0.2 ml of Yanghe decoction concentrate by gavage,the YHT+INH group was given the above two drugs by gavage at the same time,the Sham group and the TB group were given by gavage.The group was given 0.2ml normal saline by gavage for 8 weeks.At the 4th and8th week of drug intervention,4 mice in each group were sacrificed for organ weight index measurement and tissue load detection;at 8th week,the remaining 4 mice in each group were sacrificed,and spleen lymph nodes were collected.The tissue was made into single cell suspension,and the levels of IFN-?and IL-10 were detected by indirect enzyme-linked immunosorbent assay;meanwhile,the right lung tissue of each group of mice was collected for pathological analysis.Results:Comparison of organ index of mice:Comparison of lung and spleen weight index at 4 weeks and 8 weeks:Compared with Sham group,the other four groups were significantly increased;compared with TB group:INH,YHT,YHT+INH group was significantly decreased;compared with INH group Compared with the TB group,the YHT+INH group decreased;the spleen weight index:compared with the Sham group,all groups were significantly increased;compared with the TB group:the INH,YHT,and YHT+INH groups were significantly decreased;compared with the INH group,the YHT group increased;The YHT+INH group decreased significantly.The comparison between groups was lower than 4 weeks at 8weeks;comparison of the amount of bacteria in the lung tissue of mice:no bacteria were detected in the Sham group in each time period.Comparison between groups,compared with TB group:INH group and YHT+INH group were significantly lower than the TB group at the 4th and 8th week of treatment.Compared with the TB group,the YHT group had no significant difference at the 4th week;it was lower than the TB group at the 8th week;compared with the INH group:the bacterial load in the YHT group was significantly increased at the 4th week and the 8th week;there was no significant difference in the YHT+INH group.In-group comparison,compared with 4 weeks,the amount of bacteria in the lungs of the TB group increased at 8 weeks;the amount of bacteria in the lungs of the INH,YHT,and YHT+INH groups decreased at 8 weeks,and the YHT group was lower than before.The YHT+INH group showed a similar trend to the INH group;comparison of the levels of INF-?and IL-10 in mouse spleen lymphocytes:Compared with the Sham group,the expression of INF-?in the TB group was significantly increased,and the expression of INF-?in the INH group was significantly higher Compared with the TB group,the expressions of INH,YHT,and YHT+INH group were significantly decreased;compared with the INH group,the YHT group and the YHT+INH group were significantly increased;compared with the Sham group,the IL-10 expression in the TB and INH groups was significantly decreased;Compared with the TB and INH groups,the expression of IL-10 in the YHT and YHT+INH groups was significantly increased;HE staining results of the lung tissue of the mice showed that the lung tissue structure of the Sham group was normal,and there was no inflammatory cell infiltration,while the lung tissue of the TB group was normal.Inflammatory cells aggregated,mainly lymphocytes,and interstitial edema;no typical necrotizing lesions of tuberculosis were seen;compared with the TB group,the tissue inflammation in the INH group and the YHT group was milder,and the YHT+INH group had less inflammation.Cell aggregation was significantly reduced.Conclusion:Yanghe decoction combined with isoniazid can significantly improve the organ weight index of mice,promote the clearance of Mycobacterium tuberculosis,and improve the pathological damage of organs.The mechanism of action may be related to the regulation of Th1/Th2cytokine levels.(3)Yanghe decoction regulates PI3K/Akt pathway and inhibits apoptosis of mouse tuberculosis lesionsObjective:Through the H37Ra strain infection mouse model to observe whether Yanghe Decoction can inhibit the apoptosis of infected lesions by regulating the PI3K/Akt pathway,and provide a basis for Yanghe Decoction in the treatment of spinal tuberculosis.Methods:At the 8th week of drug intervention,all mice were sacrificed,the eyeballs were removed to collect blood,and the lungs of mice were dissected under aseptic operation.The levels of serum TNF-?and Bax were detected by ELISA;the m RNA expressions of PI3K,AKT,Bax and Bcl-2 in lung tissue were detected by real-time polymerase chain reaction(PCR);the PI3K,AKT,Bax and Bcl-2 m RNA expressions in lung tissue were detected by Western blotting(Westren Blot).p-AKT,AKT,Bax,Bcl-2 protein expression;TUNEL method to detect the apoptosis of lung tissue.Results:The ELISA results showed that compared with Sham,the serum levels of TNF-?and Bax in the TB group were significantly increased.Compared with the TB group,the TNF-?and Bax levels in the YHT+INH group were significantly decreased.The contents of TNF-?and Bax in the group decreased.The PCR results showed that compared with Sham,the m RNA expressions of PI3K,AKT and Bax in the lung tissue of the mice in the TB group were significantly increased,and the expression of Bcl-2m RNA was significantly decreased;The expression of Bcl-2 m RNA was significantly increased;compared with the INH group,the expression of Bax m RNA in the YHT+INH group was significantly decreased,and the expression of Bcl-2 m RNA was significantly increased;Western blot results showed that compared with the Sham group,PI3K,p-Akt and p-Akt in the lung tissue of the TB group were significantly increased.Compared with the INH group,the expression of PI3K,p-Akt and Bax protein in the lung tissue of the YHT+INH group was significantly decreased,and the expression of Bcl-2 protein was increased;TUNEL The results showed that compared with the Sham group,the apoptosis of lung tissue of the mice in the TB group was significantly increased,and compared with the TB group,the apoptosis index of the lung cells in the YHT+INH group was significantly decreased.Conclusion:Yanghe decoction can inhibit the apoptosis of infected lesions in mice,and its mechanism may be related to the PI3K/Akt signaling pathway.