Protoporphyrin ? Accumulation Induced Liver Injury After Receiving A HRZE Antitubercular Regimen

At present,China is one of the 30 countries with high burden of tuberculosis in the world,with about 900,000 new cases of tuberculosis every year,ranking the third in the world.The first-line anti-tuberculosis drugs mainly include Rifampin(RFP,R),isoniazid(INH,H),pyrazinamide(PZA,Z),ethambutol(EMB,E).They are common drugs causing drug-induced liver injury in clinical practice.At present,there are many studies on the mechanism of liver injury caused by anti-tuberculosis drugs,but no conclusion has been reached.Protoporphyrin ?,that is,free protoporphyrin,is the last intermediate produced in the heme ferrous biosynthesis pathway,It is also an endogenous hepatotoxic substance.Under physiological conditions,the biotransformation or transport of Pp? in the liver is rapid.Once the homeostasis of Pp? is disrupted,the liver becomes the damaged primary organ.We speculated that the ATLI was related to PP?.Studies have shown that the accumulation of Pp? in the liver resulting from the combination of RFP and INH is related to PXR that mediates the biosynthetic pathway of heme heme.Ferrous chelatase(FECH),the last key enzyme in the biosynthesis pathway of heme,catalyzes the implanting of ferric ions into Pp? to produce heme.FECH is the only key enzyme that can synthesize heme heme in human body.The deficiency or inhibition of this enzyme will lead to the deficiency of heme heme synthesis and the accumulation of Pp?.Objective This project is mainly to study the correlation between Pp? accumulation and liver damage caused by anti-tb drugs,and to study the mechanism of the influence of anti-tb drugs HRZE on PXR and FECH during Pp? accumulation.It can be used as a laboratory indicator of liver damage caused by anti-tuberculosis drugs and promoted for clinical use,so as to ensure the effectiveness and safety.Method The project is divided into the following two parts:Part ?.Study on the correlation between protoporphyrin ? accumulation and HRZE-related liver injury and part of the mechanism At the clinical levelCollect blood samples from TB patients,and The control group(without anti-tuberculosis drugs),treatment group 1(without ATLI after taking HRZE)and treatment group 2(with ATLI)were set up.The concentration of Pp? in blood of different groups was determined by fluorescence spectrophotometer,and the correlation between Pp?accumulation and ATLI was analyzed.The accumulation of Pp? caused by the combination of anti-tuberculosis drug HRZE is thought to be related to the activation of progesterone X receptor(PXR).CYP3A4 is the PXR receptor substrate,which can reflect the expression level of PXR.The correlation between Pp? accumulation and PXR activation was reflected by the detection of serum CYP3A4 concentration in the control group,treatment group 1 and treatment group 2.The serum concentrations of FECH and protoporphyrin zinc in TB patients in the control group,treatment group 1and treatment group 2 were detected,and the results could reflect the correlation between Pp? accumulation and FECH concentration and activity.Part ?.In vitro pregnane X receptor(PXR),chelating ferrous enzyme(FECH)in HRZE to protoporphyrin ? accumulation in mechanism researchIn addition,Hep G2 cells were treated with INH,RFP,PZA,EMB ethambutol and pyrazinamide in vitro.Western blot was used to test the protein expression of CYP3A4 in different groups,and the results could reflect the expression of PXR,proving the correlation between the accumulation of Pp? and PXR excitation.Detection of FECH concentration in vitro after HRZE dosing treatment,The results can reflect the effect of HRZE on the expression level of FECH enzyme protein.Through 5-ALA respectively combined INH,RFP and PZA,EMB dosing Pp? concentration in vitro cells after processing,the result can reflect the HRZE to FECH enzyme inhibition.In vitro FECH were given INH,RFP,PZA and EMB,FECH activity change detection in Fe2+and Pp?synthesis in the process of ferrous protoporphyrin,and respectively INH,RFP and PZA,EMB and FECH molecular docking,all prove the existence of the activity of isoniazid to FECH inhibition.Results Clinical test results show that TB patients given HRZE liver injury occurred in vivo Pp? levels after treatment,the serum concentration of CYP3A4,serum zinc protoporphyrin concentrations significantly increased(P < 0.01),the concentration of each group FECH no statistical difference(P > 0.05),illustrate ATLI is related to the accumulation of Pp?,HRZE can activate the PXR,inhibition activity of FECH exist.In vitro cell test results showed that after Hep G2 cells were treated with INH,RFP,PZA and EMB respectively,the protein expression of CYP3A4 in the RFP group was significantly increased,showing a statistical difference(P<0.01).There was no statistical difference between the other dosing groups and the normal group(P>0.05),indicating that RFP could activate PXR.There was no significant change in FECH concentration between the groups(P>0.05).In vitro Pp? synthesis increased after the addition of5-ALA,in which Pp? increased significantly in the INH+5-ALA group,and the difference was statistically significant(P<0.05).There was no significant difference between the other dosing groups and ALA group(P>0.05).In vitro FECH were given INH,RFP and PZA EMB,dosing,INH group of Fe2+ and Pp? synthesis ferrous protoporphyrin decreased significantly in the normal group(P < 0.01),the molecular docking results at the same time,indicate that INH to FECH obvious inhibitory effect.Conclusion HRZE-related liver injury is related to Pp? accumulation,and Pp? accumulation is mainly related to RFP activation of PXR and INH inhibition of FECH activity.