The Protective Effects Of Diallyl Trisulfide Against Isoniazid-and Rifampicin-Induced Hepatotoxicity

OBJECTIVEIsoniazid (INH) and rifampicin (RFP) are still the main first-line anti-T.BILI drugs in the world; the combination of INH and RFP can be effective in treating tuberculosis. However, the large number of clinical studies showed that the combination of INH and RFP could cause some patients to severe drug-induced liver damage and even liver failure, resulting in treatment interruptions and non-standard use of drugs, and then threatened patients’life and health, and leaded to drug-resistant tuberculosis, seriously impeded the prevention and control of tuberculosis. Mechanisms of INH-and RFP-induced liver injury are not yet fully clear, the majority of current studies suggested that oxidative mechanisms is the main mechanism of the damage, as well as some studies have found that INH can cause liver disorders of the immune system.Diallyl trisulfide (DATS) is a garlic product rich in antioxidant active ingredients, with a wide range of pharmacological effects. Studies have shown that DATS can be effective in preventing a variety of drugs such as alcohol, carbon tetrachloride, arsenic-induced liver damages, but the protective effect against INH-and RFP-induced liver damage has not been reported.This study by reference to relevant literature was operated as follows, Kunming mice were fed with the INH/RFP mixed solution for 1Odays to establish mouse model of INH-and RFP-reduced liver injury, and three doses of DATS were treated to study the protective effects. At the end of study, we measured serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (T.BILI) indicators, and liver histopathological to evaluate the DATS’spreventive effect against the liver damage caused by INH and RFP, while measuredthe levels of liver glutathione (GSH), malondialdehyde (MDA),the content of intrahepatic interleukin (IL-Iβ),Tumor Necrosis Factor(TNF-α) and the Kupffer cells (KCs) activation by the mouse liver immunohistochemistry and the carbon phagocytosis experiment to explore the possible antioxidant and immune mechanisms.METHODS1, Animal model120 male mice (SPF,20-22g), were randomly divided into six groups(N=20): control group, INH/RFP group, INH/RFP+DATS(10mg/kg bw) group, INH/RFP+DATS(20mg/kg bw) group, INH/RFP+DATS(40mg/kg bw) group, DATS(40mg/kg bw) group.mice in the INH&RFP+DATS groups and DATS group were treated with DATS (10mg/kg,20mg/kg,40mg/kg, and 40mg/kg bw, respectively) by gavage every day, while the mice in the control group and INH&RFP group received equal volume (0.1 ml/10g bw) of corn oil. Two hours after the DATS administration, all the animals except those in the control group and DATS (40mg/kg bw) group orally received an INH&RFP (100mg/kg and 100mg/kg bw, respectively), the control group and DATS (40mg/kg bw) group orally got an equal volume (0.2ml/10g bw) of physiological saline. After 10 days of co-administration,10 mice of each group were anesthetized at 24 hours after the last treatment. Blood was collected by eyeball extract method and centrifuged at 1500×g for 20 minutes at 4 ℃ to obtain serum. Liver was stripped and weighed. A portion of the liver was fixed in paraformaldehyde (4%) for histopathology and immunohistochemistry, while the other portion of liver tissue was quickly frozen in liquid nitrogen before storing at-80 ℃. The other 10 mice in each group were injected in India ink through tail vein (i.v.) to measure the phagocytic capacities.2, Effects of DATS on INH/RFP-Induced HepatotoxicitySerum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (T.BILI), liver index and liver histopathological examination were measured to evaluate the effect of DATS on INH/RFP-induced liver damage.3, DATS co-treatment effectively blocked INF/RFP-induced decrease of GSH and increase of MDALiver glutathione (GSH), oxidized glutathione (GSSH), total glutathione (T-GSH) content and malondialdehyde (MDA) levels were measured to investigate potential mechanisms.4, DATS co-treatment improved the function of immune induced by INH/RFPThe carbon clearance test, the level of interleukin 1-β (IL-1β), Tumor Necrosis Factor(TNF-a) and the immunohistochemistry of F4/80 marked for activated kupffer cell (KC) were measured to investigate potential mechanisms. Carbon particles phagocytosis experiments were also measured to detect mice phagocytic level5, Statistical MethodsUsing SPSS Statistics 18 statistical software for data analysis and processing, analysis using One-way ANOVA method, and comparison between the two groups, P <0.05 was considered statistically significant.RESULT1, DATS co-treatment attenuated INF/RFP-induced increase of serum ALT, AST and T.Bili levelsThe levels of serum ALT, AST and T.Bili were elevated in INH/RFP group compared to control group (P< 0.01):ALT activity increased from 45.98±6.79 to 123.5±15.89U/L, p<0.01; AST activity increased from 100.44±14.89 to 191.85±40.89U/L,p<0.05; T.Bili activity increased from 0.38±0.71μmol/L to 11.36±2.7μmol/L, p<0.01, which indicated the hepatic injury. DATS (1Omg/kg, 20mg/kg,40mg/kg, respectively) co-administration groups were ameliorated in the levels of ALT and T.Bili (P<0.05 or P<0.01), the levels of AST was also significantly refined in INH/RFP+DATS (20 and 40mg/kg, respectively) compared with INH/RFP group (P<0.01 or P<0.05),2, DATS co-treatment improved the liver histology in INH/RFP-intoxicated miceThe control and DATS group (40mg/kg bw) had normal lobular architecture with normal cell morphology and no obvious pathological state:cells closely packed in a funicular, the cytoplasm with red dye, the nucleus homogeneous with blue dye. The INH/RFP group emerged typical and obvious pathological characteristics in the portal triad region and other liver regions including loose irregular arrangement of liver cell, a large number of large, round cavity cells appeared and cell necrosis; nuclei were large and deep dye, central venous blood stasis. Compared with INH/RFP group, INH/RFP+DATS (l0mg/kg bw) had a small amount of small vacuoles cells. But in the INH/RFP+DATS (20 and 40mg/kg bw) group, the hapatic injury were obviously recovered.3, DATS co-treatment effectively blocked INF/RFP-induced decrease of GSH and increase of MDAThe levels of T-GSH, GSH in INH/RFP had a significant reduction in 31% (P<0.05),55%(P<0.01) compared with control group. The contents of GSSH were higher than control group up to 103%(P<0.01), but this damage were well reversed by co-administration of DATS (10,20 and 40mg/kg bw; respectively). The levels of T-GSH were increased by 17%,20%,45%(P<0.05), respectively. The levels of GSH were increased by 61%,76%,133%(P<0.01), respectively. And the levels of GSSH were declined by 26%(P<0.05),38%(P<0.05),40%(P<0.01), respectively.The levels of MDA are an index of the intensity of lipid peroxidation damage in the liver. The results showed that INH/RFP group had a higher increase of 164%(P<0.01) than control group, while with the DATS co-administration, the INH/RFP+DATS (10, 20 and 40 mg/kg bw, respectively) groups were significantly decreased the content of MDA by 47%(P<0.05),56%(P<0.01),59%(P<0.01) in comparison with INH/RFP group, respectively.4, DATS co-treatment improved the phagocytic capacities by the assay of carbon clearanceThe assay of the carbon clearance test in INH/RFP group was decreased by 39% contrast to the control group, and the results showed that DATS (10,20,40 mg/kg bw, respectively) co-administration improved the carbon phagocytic capacities by 62%, 67%,79%(P<0.01) contrast with the INH&RFP group.5, DATS co-treatment led to the activation percent of the kupffer cellsThe immune staining for F4/80 of KCs in the liver showed that a reduction population of activated KCs even despaired in INH/RFP group (Fig 5.). With co-administration of DATS, the KCs in INH/RFP+DATS (lOmg/kg bw), INH/RFP+DATS (20 mg/kg bw) and INH/RFP+DATS (40 mg/kg bw) group were in varying degrees of activations than INH/RFP group, and the activations were increased with the increasing dose of DATS.6, DATS co-treatment contributed to the normal secretion of IL-ip and TNF-aThe secretions of IL-Iβ which was mainly secreted by KCs in the liver were decreased by 39% in INH/RFP group contrast with control group (P<0.01). By the DATS (10,20,40 mg/kg bw, respectively) co-treatments, the secretions of IL-Iβ were similar with the activation of KCs and was increased by 52%(P<0.05),82%(P<0.01), 113%(P<0.01), respectively.The secretions of TNF-a which was the main indicator of inflammatory response were increased by 20% in INH/RFP group contrast with control group (P<0.01). By the DATS (10,20,40 mg/kg bw, respectively) co-treatments, the secretions of TNF-a were decreased by 6%,13%(P<0.05),16%(P<0.05), respectively.CONCLUSION1, DATS could protect the liver away from the damage caused by isoniazid and rifampicin in mice.2, the mechanism of DATS against isoniazid and rifampicin induced liver injury in mice might be related to antioxidant mechanism3, the mechanism of DATS against isoniazid and rifampicin induced liver injury in mice might be related to improve the ability of KCs and inhibit the inflammatory response.