Study On The Relationship Between Baseline VD Nutritional Status And The Risk Of Anti-tuberculosis Drug Induced Liver Injury And The Possible Mechanism

Background and Objective:The first-line anti-tuberculosis drugs such as isoniazid and rifampin are effective for treating tuberculosis(TB).However,the drugs also cause various side effects to patients.Liver injury is the most common side effect of anti-tuberculosis drugs.Patients will have to stop using the drug or change the treatment regimen once liver injury occurs,which may lead to an increased risk of treatment failure and the emergence of drug-resistant tuberculosis.The mechanism of liver injury caused by anti-tuberculosis drugs is still not clear,and may be related to the elevated oxidative stress and mitochondrial damage in liver cells induced by reactive metabolites of drugs.Diabetes mellitus(DM)increases the risk of active tuberculosis,and the double burden of TB and DM has attracted more and more attention in recent years.We found that DM is an independent risk factor for liver injury in patients with tuberculosis over 45 years old in a previous study.It has been shown that vitamin D(VD)deficiency is associated with an increased risk of alcoholic and non-alcoholic liver injury.However,there are few reports about the relationship between the VD status and the risk of anti-tuberculosis drug-induced liver injury(ATLI).We found that VD supplementation can improve the liver injury induced by anti-tuberculosis drugs by reducing the level of oxidative stress and inflammation in another study.However,whether VD supplementation can improve the liver injury of DM animals caused by anti-tuberculosis drugs is still unclear.Therefore,we collected demographic and clinical data of hospitalized tuberculosis patients in Qingdao,analyzed the relationship between serum VD status and the risk of ATLI,and further explored the effect of DM on the association(Chapter 1).We establish a liver injury model induced by anti-tuberculosis drugs in DM mice,and treatment with different doses of VD to explore the protective effect and mechanism of VD on Isoniazid(INH)combined with Rifampin(RFP)-induced liver injury(Chapter 2),in order to provide evidence for clinical prevention of liver injury.Methods:The method of prospective cohort study was used in chapter one.Patients who were diagnosed with TB with and without DM in Qingdao Chest Hospital from January 2017 to December 2019 were enrolled.A questionnaire was used to collect the demographic characteristics,lifestyle and behavior habits of the participants,and physical measurements and clinical medical records were collected.At the same time the patient’s peripheral fast blood when admitted to the hospital were collected and serum 25(OH)D concentration was determined.We followed up the patients to observe the occurrence of liver injury during their treatment.The standards recommended by the US Institute of Medicine(IOM)was adopted to determine VD status,and univariate and multivariate logistic regression analysis was used to explore the relationship between VD status and the risk of ATLI.Receiver operator characteristic curve(ROC)was used to calculate the area under the curve for estimating the predictive value of the VD status in the occurrence of liver injury.We adopted the method of animal experiment in chapter two.DM mouse model was established by feeding with high-sugar and high-fat feed and injection with streptozotocin.Then the mice were randomly divided into 6 groups including control group,model group,and VD low-dose group,VD medium-dose group,VD high-dose group and positive control group(Metformin,MTF),with 15 mice in each group.All mice were given intragastric administration twice a day for 28 days.Mice of control group and model group were given0.5%sodium carboxymethyl cellulose solution for the first time.Mice of the VD low-,medium-and high-dose groups were given 130IU/kg,260IU/kg,520IU/kg VD,respectively.Mice of positive group was given 200mg/kg metformin.Two hours later,the control group was given 0.5%sodium carboxymethyl cellulose solution,and the other groups were given100 mg/kg isoniazid and 150 mg/kg rifampicin.After the last administration,the mice were fasted for 12 hours and then weighed,anesthetized,and blood was removed from the eyeballs.The serum was separated,liver,kidney and other organs were dissected and stored at-80°C.An automatic biochemical analyzer was used to analyze the serum levels of liver injury markers such as alanine aminotransferase(ALT),aspartate aminotransferase(AST).HE staining was used to observe the pathological changes of liver tissues.Enzyme-linked immunosorbent assay(ELISA)was used to determine the content of serum 25(OH)D.The colorimetric method was used to determine the activity of glutathione(GSH),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)and the content of malondialdehyde(MDA)of livers.Real-time quantitative PCR was used to detect liver mitochondrial DNA copy number.Fluorescent probe method to detect liver mitochondrial membrane potential.Western-blot was used to detect the expression of Complex I～V.Results:A total of 461 participants were included in this study,of which 48 patients occured liver injury during treatment,and the incidence of liver injury was 10.41%.The proportion of severely VD deficient in patients with liver injury was significantly higher than that in those without liver injury(66.7%vs.45.3%,p=0.005).After adjusting for age,BMI,alcohol consumption,and DM,the risk of liver injury in patients with severely VD deficiency was nearly three times than other patients(OR=2.91,95%CI:1.49-5.69).Severely VD deficiency combined with age,BMI,hypoproteinemia,DM and alcohol consumption have a certain predictive effect on liver injury(AUC=0.74,95%CI:0.67-0.81).According to the stratified analysis based on whether combined with DM and the results showed that after adjusting for related confounding factors,severely VD deficiency increased the risk of liver injury in patients with tuberculosis only and PTB-DM by 1.9 times(OR=2.92,95%CI:1.21-7.06)and2.1 times(OR=3.11,95%CI:1.07-9.07),respectively.A stratified analysis according to age showed that severely VD deficiency increased the risk of liver injury in patients with the age over 45 years by about 4 times(OR=5.13,95%CI:1.94-13.57).Stratified analysis based on BMI showed that severely VD deficiency increased the risk of liver injury in patients with BMI≥18.5 kg/m~2 by 2.3 times(OR=3.33,95%CI:1.44-7.71).The results of animal experiments in Chapter 2 showed that:(1)Compared with the control group,the weight of the model group was significantly reduced when intervene for 4weeks(p<0.05),and there was no significant difference in the weight of the mice among the other groups.(2)Serum 25(OH)D level of the model group was significantly lower than that of the control group,and significantly increased in the medium-and high-dose VD intervention group(p<0.05)(3)Serum ALT,AST,ALP,TBIL,DBIL and TBA in the model group were significantly higher than those in the control group(p<0.05);in the medium-and high-dose VD intervention group,serum ALT,AST,ALP and DBIL were significantly decreased compared with model group(p<0.05).(4)In the model group,pathological changes such as disorder of liver cell arrangement,inflammatory cell infiltration,and ballooning were clearly observed.VD intervention attenuated the pathological changes of liver fat deformation and hepatocyte necrosis.(5)The GSH-Px and SOD activity in the liver of the model group was significantly lower than that in the DM group(p<0.05),and the MDA content was significantly higher(p<0.05).The activity of GSH-Px and SOD in the medium-and high-dose VD intervene group significantly improved(p<0.05),and the MDA content was significantly decreased(p<0.05).(6)The mitochondrial membrane potential of the model group was significantly lower than that of the control group,and the mitochondrial membrane potential of the liver of mice in the VD medium and high-dose intervention group was significantly increased(p<0.05).(7)The mitochondrial DNA copy number in the model group was lower than that in the control group,but the difference was not statistically significant.Compared with the model group,VD intervention can significantly increase the mitochondrial DNA copy number(p<0.05).(8)Compared with the control group,the expression of Complex II in the liver of the model group was significantly decreased(p<0.05),and VD intervention was not found to have a significant regulatory effect on ComplexesⅠ～Ⅴ.Conclusion:Severely VD deficiency is an independent risk factor for drug-induced liver injury in pulmonary tuberculosis patients,and when diabetes is combined,the association between VD status and ATLI is stronger.Severely VD deficiency combined with age,BMI,hypoproteinemia,alcohol consumption and DM prevalence may have certain predictive value for ATLI.VD supplementation improves the liver injury of diabetic mice induced by INH combined with RFP by reducing the level of oxidative stress,inhibiting the decrease of mitochondrial membrane potential and maintaining the stable of mitochondrial DNA copy number.