Mechanism Of Protoporphyrin Ⅸ Accumulation In Isoniazid And Rifampicin Induced Liver Injury

Isoniazid and rifampicin are recommended by the world health organization for the first-line anti-tuberculosis drugs,which have the advantages of high efficacy,convenient oral administration and low price.However,the co-therapy of isoniazid and rifampicin or alone has hepatotoxicity,and long-term use may result in drug-induced liver injury.Currently,there are many studies on the toxicity of isoniazid and rifampicin,but mechanistic hypothesis of INH-induced hepatotoxicity was not entirely understood.Protoporphyrin IX is an endogenous hepatotoxic substance.Large amounts of PPIX are hepatotoxic,damaging both hepatocytes and cholangiocytes.Our study confirmed that isoniazid and rifampicin caused hepatotoxicity and the accumulation of protoporphyrin IX.The combination can aggravate protoporphyrin IX accumulation.The accumulation of protoporphyrin IX is relevant to activation of pregnane X receptor(PXR,pregnane X receptor).Ferrochelatase(FECH)is the terminal enzyme of the heme biosynthetic pathway,responsible for catalyzing the insertion of ferrous ion into protoporphyrin IX.Any loss of activity of this enzyme might result in intracellular heme deficiency and accumulation of protoporphyrin IX.We demonstrated that isoniazid could inhibit FECH enzymatic activity directly in vivo and vitro.It is speculated that inhibition of the FECH activity is responsible for INH-induced liver injury,which contribute to its PPIX accumulation function.This study provides novel insight into the mechanism of INH-induced liver injury.This study is divided into two parts.Part I.The study of progesterone X receptor mediated isoniazid and rifampicin hepatotoxicity.Through the establishment of isoniazid and rifampicin-treatment animal model of C57 BL / 6 wild type mice.We proved that the isoniazid and rifampicin alone can caused liver injury and induced protoporphyrin IX accumulation in the liver,co-therapy of isoniazid and rifampicin can aggravate protoporphyrin IX accumulation,further caused liver damage.From two aspects to investigate in vitro protoporphyrin IX accumulation and activation of PXR relations,we use rifampicin of large doses with intraperitoneal injection to activate m PXR.Hep G2 cells treated with isoniazid and rifampicin in vitro.The expression of CYP3A4 protein was tested by western blot method,and the expression of PXR was reflected.The results showed that the accumulation of protoporphyrin IX was positively correlated with PXR activation.Part II.Isoniazid induced liver injury induced by the inhibition of ferrous chelatase.An animal model of isoniazid induced liver injury induced by isoniazid in kunming was established.It was proved that isoniazid gavage could induce liver injury in mice significantly,and the accumulation of protoporphyrin IX in mice liver was increased in dose and time dependent.We used high performance liquid chromatography(HPLC)method to detect the contents of zinc protoporphyrin judgement chelating ferrous enzyme activity.Isoniazid in fluorescent microscope observed the protoporphyrin IX fluorescence method in vitro.It proved that ferrochelatase significantly inhibit enzyme activity in vitro and vivo.The combination of isoniazid and ferrochelatase dimer was demonstrated by the computer structure docking simulation,which further proved that isoniazid itself had an inhibitory effect on ferrochelatase.The direct inhibition of isoniazid to the activity of ferrochelatase in the liver,resulting in the accumulation of the protoporphyrin IX in the liver may be the cause of hepatic injury caused by isoniazid.