HEPATOCYTE-DERIVED MICRORNAS AS BIOMARKERS OF HEPATIC INJURY IN ISONIAZID-INDUCED HEPATOTOXICITY

Study aimed to evaluate the diagnostic value of hepatocyte-derived microRNAs and their genes, MDA, GSH and GPX in experimental rat model of liver damage induced by isoniazid. In this study, 72 adult male rats (130-150 gm) were divided into six groups and given treatments for 21 days: control group:(1 ml saline /Kgb.wt/day I/P).INH group: 100 mgINH/kgb.wt/dayI/P. INH + Rutingroup: 100 mg INH/kg b.wt/day I/Pwith 200 mgrutin/kg b.wtorally. INH + NAC group: 100 mg INH /kg b.wt/day I/Pwith 300 mgNAC/kg/day b.wt orally. Rutingroup: 200 mgrutin/kg/day orally.NAC group: 300 mg NAC /kg b.wt orally. Tissue samples were obtained after 14 and 21 days foranti-oxidants as well as miRNAs 122 and 125b, CyclinG and STAT3 detection. Isoniazid injection into male rats induced a significant upregulation of CG1and STAT3 expression levelconcomitant with a downregulation of miRNA122 and miRNA125b.Meanwhile, ratstreated with rutin or NAC combined with INH developed a downregulation of CG1gene and STAT3 expression levelwithupregulation of miRNA122 and miRNA125b. INH induced a significant increase in MDA levelwith a significant decrease in GSH concentration and GPx activity. While INHinjection with rutin or NAC treatment induced asignificant decrease in MDA level with a significant increase in GSH concentration and GPx activity.Taken together these findings suggest that hepatocyte-derived microRNAs 122 and 125bcould beused as early sensitive biomarkers for hepatotoxicity inducedduring isoniazid treatment.

Study aimed to evaluate the diagnostic value of hepatocyte-derived microRNAs and their genes, MDA, GSH and GPX in experimental rat model of liver damage induced by isoniazid. In this study, 72 adult male rats (130-150 gm) were divided into six groups and given treatments for 21 days: control group:(1 ml saline /Kgb.wt/day I/P).INH group: 100 mgINH/kgb.wt/dayI/P. INH + Rutingroup: 100 mg INH/kg b.wt/day I/Pwith 200 mgrutin/kg b.wtorally. INH + NAC group: 100 mg INH /kg b.wt/day I/Pwith 300 mgNAC/kg/day b.wt orally. Rutingroup: 200 mgrutin/kg/day orally.NAC group: 300 mg NAC /kg b.wt orally. Tissue samples were obtained after 14 and 21 days foranti-oxidants as well as miRNAs 122 and 125b, CyclinG and STAT3 detection. Isoniazid injection into male rats induced a significant upregulation of CG1and STAT3 expression levelconcomitant with a downregulation of miRNA122 and miRNA125b.Meanwhile, ratstreated with rutin or NAC combined with INH developed a downregulation of CG1gene and STAT3 expression levelwithupregulation of miRNA122 and miRNA125b. INH induced a significant increase in MDA levelwith a significant decrease in GSH concentration and GPx activity. While INHinjection with rutin or NAC treatment induced asignificant decrease in MDA level with a significant increase in GSH concentration and GPx activity.Taken together these findings suggest that hepatocyte-derived microRNAs 122 and 125bcould beused as early sensitive biomarkers for hepatotoxicity inducedduring isoniazid treatment.

…………………………………………………………………………………………………….... Introduction:-
MicroRNAs are small non-coding RNAs first described by (Lee, Feinbaum et al. 1993). They are approximately 20 -27 nucleotides long (Engels and Hutvagner 2006).Hepatocyte derived miRNAs highly stable and sensitive blood based biomarkers for hepatocellular injury in animal models and in human patients with normal and high ALT activities (Laterza, Lim et al. 2009, Wang, Zhang et al. 2009). Drug-induced liver injury is a major health problem that challenges not only health care professionals but also the pharmaceutical industry and drug regulatory agencies (Yuan and Kaplowitz 2013). The drug-induced injury could be induced through different ways including direct toxic effect; immunological reaction or active metabolite that is formed by the drug (Bayram, Ozogul et al. 2005).Isoniazid is a widely used and effective first-line agent for treatment of tuberculosis; the most important clinical adverse reaction of INH is anti-tuberculosis drug-induced liver injury (ADLI) (Jaswal, Sinha et al.

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MiRNA 122 and 125b: MiRNA extraction: mirVana PARIS kits (Ambion, Life Technologies, USA, #AM1556) To isolate miRNA from liver tissues, samples are homogenized in Lysis Buffer. The lysate is then mixed with 25% ethanol. Mixture is passed through a glass-fiber filter, large RNAs are immobilized. The ethanol concentration of the filtrate is then increased to 55%, and it is passed through a second filter where the small RNAs become immobilized, washed a few times, and eluted. cDNA synthesis Quanti-Mir RT kit (SBI, System Biosciences, Cat. # RA420A-1) This kit depends on the poly(A) method. In this method, a poly(A) tail is added to the 3' end of each mature miRNA done by poly (A) polymerase. Tailed miRNAs are then subjected to RT using a universal RT primer containing 2 to 3 degenerate nucleotides at 3' end followed by an oligo and universal reverse primer sequence. The synthesized cDNA is amplified with specific forward and universal reverse primers.

Real time PCR:
Real-time PCR with SYBR Green was used to measure expression of miRNA122 and miRNA125b in the liver tissues, with miRNA16 as an internal reference Table (2).The isolated cDNA were amplified using 2X Maxima SYBR Green/ROX qPCR Master Mix following the manufacturer protocol (Thermo scientific, USA, # K0221).

Oxidants and anti-oxidants parameters:
MDA, GSH and GPxwere measured according to manufacturerʼs instructions.

Statistical analysis:
Statistical analysis was performed using the statistical software package SPSS for windows Version 19. ANOVAtest was used to determine significant differences between experimental groups followed by Duncan. Results was expressed as the mean ± standard error of mean.

Results:-
The present study showed that miRNA122 as well as miRNA125b expression levels in liver tissues were significantly reduced inisoniazid group at 14 and 21 days as compared to the control group (Tables 3 and 4). This downregulation of miRNA122 as well as miRNA125b expression levels was significantly upregulated following coadministration of isoniazid with rutin or N-acetyl cysteine. However, rutin or N-acetyl cysteine administrated groups did not change miRNA122expression level in rat liver.
The obtained results revealed a significant upregulation of CG1and STAT 3genes expression level in liver tissues of isoniazid groupas compared to control group (Table 5 and 6). This elevated expression was significantly downregulated following co-administration of isoniazid with rutinorN-acetyl cysteine. Results showed significant downregulation in isoniazid group treated with NAC compared with isoniazid group treated with rutin. Moreover, non-significant difference was noticed among rutin and NAC administrated groups compared with control group.
Isoniazid group showed significant increase in MDA level while, there was significant decrease in GSH concentration and GPxactivity when compared with control group (Table 7).
In comparison to isoniazid group, Isoniazid injection withrutin or N-acetyl cysteinetreatment, there wasa significant decrease in MDA level concomitant with a significant increase in the GSH concentrationand GPx activitybut did not reach that of control.
It was noticed that rutin group showed significant decrease in MDA level while, there were significant increase in GSH concentration and GPxactivity compared with control, Meanwhile, N-acetyl cysteine group showed nonsignificant changes in MDAlevel, GSHconcentration and GPxactivity compared with control.

Discussion:-
MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate post-transcriptional gene expression (Bartel 2009). Regarding to molecular analysis, Isoniazidinjection exhibited significant downregulation in 373 miRNA122 and miR-125bwhile there wasupregulation in cyclin G1 and STAT 3 genes at 14 and 21 days when compared with control group. Our results agree with Li, (2018).Hypermethylationof CpG islands in the promoter region of miRNAgenes is one of most important mechanisms that inducemiRNA expression downregulation (Lynch, 2016). Since miR-122 is the most abundant miRNA in the liver and its expression in the liver wasdecreased in advanced liver diseases, such as primary biliary cirrhosis (PBC) and hepatocellular carcinoma (HCC). Mice treated with CCl4 for 6 weeks causes liver fibrosis that led to significant downregulation of miR-122 in mouse liver. This result was similar to that observed in human patients with primary biliary cirrhosis (Esau, 2006).Also, Expression level of miR-122 decreased significantly in activated HSCs, which contribute to the progression of liver fibrosis throughupregulating the expression of prolyl 4-hydroxylase and the subsequent production of overlycrosslinked collagen. It should be noted that miR-122 is downregulated in the livers of patients with primary biliary cirrhosis (Padgett, 2009) as well as in the livers of mice treated with CCl4 (Marquez, 2010). Moreover, cell cycle protein G1(Cyclin G1) and cationic amino acid transporter-1 (CAT-1) which are miR-122 targets gene also increased at the mRNA and protein levels,which suggests that downregulation of miR-122 due to the upregulation of Cyclin G1 and CAT-1 and might play a role in INH induced hepatic injury (Li, 2018). On the other hand, MiR-125bis one of miRNAs identified to be associated with inflammation (Li, 2018).Also, STAT3 the common target gene of miR-125b (Carraro, 2009). STAT3 is an important marker in the differentiation of T-helper cells and regulates the transcription of inflammatory genes (Harris, 2007).T-helper cells mainlysecrete pro-inflammatory cytokine IL-17 (McGeachy, 2008) which stimulate the activation of various pro-inflammatory cytokines and chemokines and participate in drug-induced liver injury (Wang, 2014). TNF-α and IL-6 are key proinflammatorycytokines that initiate the inflammatory response and induce massive hepatocyte apoptosis. Also, liver can activate Kupffer cells to release inflammatory cytokines (IL-6 and TNF-α) when stimulated by toxic substances (Yano, 2012)so STAT3 can be activatedin hepatocytes following TNF-α and IL-6 stimulation then STAT3 activation plays an important role in cell survival, differentiation, transformation, apoptosis and inflammation (Bromberg, 2000).
Isoniazid injection with rutin treatment showed significant upregulation in MiRNA 122 and MiRNA 125b but showed significant downregulation in cyclin G1 and STAT 3 genes at 14 and 21 days compared with isoniazid group. Cyclin G1 downregulation may be due to anti-inflammatory effect of rutin by downregulating IL-6 and TGF-1, which promotes extracellular matrix deposition and fibrosis (Edranov, 2012). Also, rutin produced antiinflammatory effects by inhibiting pro-inflammatory cytokines in adjuvant-induced arthritis in rats (Kauss, 2008).MiRNA 122 upregulation may be due to downregulation of TGF-1 and Cyclin G1.Moreover, Rutin inhibits inflammatory responses in ultraviolet-irradiated mouse by preventing the phosphorylated STAT3 levels (Choi, 2014).This indicates that rutin play an important role in upregulation of MiRNA 125b and inprotection against isoniazid induced hepatotoxicity.
Isoniazid injection withN-acetyl cysteine treatmentshowed significant upregulation in MiRNA 122 and MiRNA 125b with significant downregulation in cyclin G1 and STAT 3 genes at 14 and 21 days when compared with isoniazid group. Studies have shown that activation of thearyl hydrocarbon receptors increases expression of CD36, a scavenger receptor involved in fatty acid uptake, andtumor necrosis factor alpha (TNFα), a mediator of inflammation (Lee, 2010, Vondracek, 2011. Both genes have been implicated in liver steatosis (Greco, 2008).Since,NAC possible targets genes areCD36 and TNFα, so it has beenreduced both liver CD36 and TNFα expression in mouse non-alcoholic steatohepatitis (NASH) (Baumgardner, 2008) andinhibition of TNF is regarded as a therapy to block fatty liver and relieve liver injury (Feagins, 2015).Moreover,NAC is an effective hepatic antioxidant that inhibited non-alcoholic steatohepatitis induced lipid peroxidation, increased hepatic GSH, blocked non-alcoholic steatohepatitis autoimmune responses, inhibited production of TNFα, and attenuated inflammation leading to reduction in cellular damage, hepatocyte injury, and fibrosis (Baumgardner, 2008). Isoniazid combined with NAC caused significant decrease in MDA level and significant increase in GSH concentration and GPxactivity compared with INH. Our results agree with Eşrefoğlu, (2006). NAC has been tried in the treatment of various liver injuries because of its known antioxidant properties (Baniasadi, Eftekhari et al. 2010). NAC exerts its antioxidant action by facilitating glutathione biosynthesis and scavenging the ROS formed during oxidative stress (Ocal, Avlan et al. 2004).

Conclusion:-
Isoniazid induced downregulation expression of microRNAs (122 and 125b) and upregulation of their genes (Cyclin G and STAT3). Therefore, hepatic microRNAs (122 and 125b) aresensitive biomarkers in INH hepatic injury. Moreover, N.acetylcysteine and rutin has hepatoprotective effect due to their anti-oxidant effects which alleviate isoniazid hepatic injury.Also, N.acetylcysteineis more potent than rutin in liver injury treatment.