Suppression of iNOS/HIF-1α/MMP-9/α-SMA/collagen Axis of Fibrosis and Systemic Hypertension in Thioacetamide-induced Liver Injury by Resveratrol

Chronic liver injury can lead to hepatic failure and the only available method of treatment would be liver transplantation. The link between nitrosative stress (iNOS), hypoxia-inducible factor-1α (HIF-1α), and alpha-smooth muscle actin (α-SMA), in thioacetamide (TAA)-induced liver brosis and hypertension in treatment with the anti-inammatory and antioxidant, resveratrol (RES) was not investigated before. Consequently, we injected rats with either 200 mg/kg TAA for 8 weeks starting at week 2 (model group) or pretreated them before TAA injections with RES (20mg/kg) for two weeks and continued on RES and TAA until being culled at week 10 (protective group). In the model group, we documented the induction of hepatic brosis and upregulation of tissue iNOS, HIF-1α, and the pro-brotic biomarkers α-SMA and matrix metalloproteinase-9 (MMP-9) that was signicantly (p ≤ 0.0014) ameliorated by RES. RES also signicantly (p ≤ 0.0232) reduced triglycerides (TG), cholesterol (CHOL), very low-density lipoprotein (vLDL-C), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart rate (HR) induction by TAA. Also, a signicant (p<0.0001) positive correlation between iNOS/HIF-1α/α-SMA/collagen axis and hypertension and liver injury biomarkers was observed. These ndings indicate that the hepatotoxic compound, TAA augments iNOS/HIF-1α/MMP-9/α-SMA/collagen mediated brosis and hypertension, and is inhibited by RES for 10 weeks. immunostaining above exemplied in histograms (D). Liver lysates immunoblotted with antibodies against HIF-1α (E and inset), and b-actin as a loading control (lower bands, inset


Introduction
Chronic hepatic disease complications represent a major encounter to the health sector including liver transplantation caused by liver failure (Neff et al., 2011). TAA is a severe hepatotoxic agent that causes liver brosis(Al-Hashem et al., 2018), cirrhosis and liver cancer (De Minicis et al., 2013), which depends on the length of exposure of the body to this agent. Animals injected with TAA for 6 to 10 weeks caused liver brosis and cirrhosis (Wallace et al., 2015). Chronic liver insults such as toxins, chemicals, alcohol abuse, viruses, cholestasis, and autoimmune diseases have been associated with the pathophysiology of hepatic brosis that could also lead to liver cirrhosis and ultimately liver failure (Friedman, 2003, Czaja, 2014. Therefore, the prevention of hepatic brosis development is a very valuable target aimed by the clinician to prevent the advancement of liver cirrhosis and hepatic failure. Hepatic stellate cells (HSCs) activation with these insults is the main driver of liver brosis since HSCs produce most of the brogenic extracellular matrix upon activation (Gressner and Weiskirchen, 2006). Induction of HIF-1α, and biomarkers of pro brogenesis, α-SMA and collagen type III by carbon tetrachloride (CCl4) in rats promote liver brosis via the activation of HSCs (Zhao et al., 2014). In addition, HIF-1α induced pulmonary hypertension and brosis (Ball et al., 2014). TAA is reported to activate HIF-1α and α-SMA in HSCs cell lines and animal models of liver disease (Al-Hashem et al., 2018). Furthermore, oxidative stress and in ammation are involved in promoting brosis by HSCs and in TAA hepatic intoxication (Robert et al., 2016). Overproduction of reactive nitrogen species (nitrosative stress; iNOS induction) also plays a role in liver disease pathologies such as CCl4-and cholesterol-induced hepatic brosis (Anavi et al., 2015, Yu et al., 2019. Indeed, in animal models of cholesterol-induced liver brosis, iNOS de cient mice developed less liver brosis compared with wild type mice that showed a greater brotic tissue and upregulation of HIF-1α and MMP-9 (Anavi et al. 2015).
RES, a plant phytoalexin, is widely used in research for cardiovascular and kidney protection (Chen et al., 2019, Zhao et al., 2018. It also inhibits preeclampsia biomarkers (Cudmore et al., 2012, Al-Ani, 2013, and prevents apoptosis and promotes cell survival (Alayev et al., 2014). Also, numerous types of liver diseases such as sinusoidal obstruction syndrome and hepatic steatosis (Trepiana et al., 2018) were inhibited by RES as well as HSCs (Kawada et al., 1998). Furthermore, in a mouse model of CCl4-induced liver brosis, RES ameliorated brosis, which was associated with the inhibition of iNOS(Yu et al., 2019). Therefore, these reports prompted us to speculate the activation of iNOS/HIF-1α/MMP-9/α-SMA mediated liver brosis and hypertension by TAA which could be inhibited by RES.

Materials And Methods
Animals 24 rat male Albino rats weighing 180-200 gm were included in the experiment. The study follows the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). And approved by the Ethical Committee at King Khalid University, Rats were housed at a controlled temperature (25 ± 2°C) and relative humidity(50 ± 10% ), with twelve-h light/twelve-h dark cycles, and had free access to food and water.

Experimental Design
One week after adaptation, 24 rats were separated into 4 groups (n= 6 per group). Firstly, the control group (Control) of rats that were non-treated and injected intraperitoneally (i.p.) with the vehicle. Secondly, the resveratrol control group (RES) of rats treated with RES suspension (20 mg/kg, orally) daily for ten weeks. Thirdly, the model group (TAA) of rats that were subjected to i.p. injections with TAA (200 mg/kg, twice per week) for eight weeks (starting at week 3)(Wallace et al., 2015). Fourthly, the protective group (RES+TAA) that includes rats given RES as above from day one till the end of the experiment, at the 10th week, and received TAA as above for eight weeks. After the completion of the experiment, blood samples were collected under anaesthesia using sodium thiopental (40 mg/kg), and animals were killed by cervical dislocation, and liver tissue specimens were harvested.

Histological Examination
Harvested liver specimens were xed overnight in ten % formalin and then dehydrated with ascending grade of alcohols. Para n blocks were prepared by the standard method, and 5µm thick sections were de-para nized and rehydrated. Hepatic sections were then stained with Masson's trichrome to assess the degree of hepatic brosis.
Immunohistochemistry of iNOS and α-SMA Immunohistochemical staining was performed using anti-inducible nitric oxide synthase (iNOS) (Abcam, cat # ab15323) and anti-alpha-smooth muscle actin (α-SMA) (Dako; cat # M0851) as a marker for HSCs activation. Antigen retrieval was conducted, followed by the application of the primary antibody overnight in a humidity chamber and the secondary antibody for 30 minutes. Sections were co-stained with Meyer hematoxylin.
Quantitative real-time polymerase chain reaction (qRT-PCR) of MMP-9 Total RNA was isolated from rats' livers using the RNeasy Mini Kit (Qiagen Pty, Victoria, Australia) and 1 µg RNA was reverse-transcribed with the complementary DNA (cDNA) synthesis kit (Fermentas, USA). Triplicate cDNA samples and standards were ampli ed in Master Mix containing SYBR green (Thermo Assessment of ALT, AST, triglyceride, cholesterol, vLDL-C, and HDL-C levels Enzymatic kits (Randox Laboratories, UK) were used to determine the blood levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The blood levels of triglycerides (TG), cholesterol (CHOL), very low-density lipoprotein (vLDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured according to the manufacturer's instructions using the commercial kits supplied by SPINREACT, Spain.
Determination of blood pressure and heart rate SBP, DBP, and MAP were measured from the conscious rats ails using the tail-cuff technique (BP monitor, LE 5001, LETICIA scienti c Instruments, Spain. Rats were warmed for half an hour at 28°C in a thermostatically controlled heating cabinet (Ugo Basile, Italy) ) for better detection of the tail artery pulse. The tail was passed through a cuff sensor that was connected to an ampli er (LE 5001, LETICIA scienti c Instruments, Spain). The cuff was attached to a sphygmomanometer and BP and heart rate was recorded on a chart, and the averages of three measurements were taken.

Statistical Analysis and Morphometry
Analyses of data were conducted utilizing SPSS with version 10.0 (SPSS, Inc., Chicago, Ill., USA). Statistical comparisons of data were performed using one-way ANOVA followed by Tukey's post hoc test. To detect a probable signi cance between two different parameters, Pearson correlation was performed.
p ≤ 0.05 was considered statistically signi cant.
Morphometry of the percentage areas of collagen deposition in Masson's trichrome stained sections and the percentage areas of α-SMA and iNOS positive immunostaining were done using "Leica Qwin 500 C" image analyzer (Cambridge, UK) in 10 non-overlapping elds for each group. Data were also analyzed using analysis of variance (ANOVA) as described above.

Results
Resveratrol (RES) inhibits TAA-induced nitrosative stress (iNOS) and hypoxia (HIF-1α) in Liver Tissues Nitrosative stress and hypoxia are known inducible factors of liver brosis (Iwakiri, 2015, Roth andCopple, 2015). To test the hypothesis that chronic TAA toxicity can augment tissue levels of nitrosative stress and hypoxia biomarkers and whether RES can protect against the induction of these parameters, we assessed the liver tissue levels of iNOS ( Figure 1A-D) and HIF-1α ( Figure 1E) protein in all rats by the end of the experiment. Immunohistochemical analysis of iNOS from liver sections prepared from the control rats showed negative staining ( Figure 1A). Whereas, many iNOS positive cells were depicted in liver sections of the TAA group ( Figure 1B), which were substantially but not completely inhibited by resveratrol in the RES + TAA group ( Figure 1C). The mean area % of iNOS immunostaining in the liver sections of all groups is shown in Figure 1D.
Western blots analysis of hepatic tissue homogenates prepared from the model group (TAA) revealed an increase in HIF-1α protein expression that was signi cantly (p < 0.0001) ameliorated by RES ( Figure 1E). Conversely, the inhibition of HIF-1α by RES was signi cantly (p < 0.0001) elevated compared with the control rats, which denotes a complete inhibition by RES was not achieved. Also, there is a signi cant (p<0.0001) correlation (r = 0.949) between iNOS score and HIF-1α ( Figure 1F).

Resveratrol (RES) decreases TAA-induced biomarkers of liver brosis α-SMA and MMP-9 in injured liver
α-SMA is a well-known pro-brogenic marker (Carpino et al., 2005) and MMP-9 is now considered as a pro-brotic and not anti-brotic marker as previously thought (Murthy et al., 2010). Therefore, we evaluated their tissue levels in the TAA and treated group using immunohistochemistry and qRT-PCR analyses ( Figure 2). Using immunohistochemical analysis, hepatic tissue sections of the control group revealed few areas of α-SMA positive cells in the hepatic vascular endothelium (Figure 2A). Many α-SMA positive cells were illustrated in liver sections of the model (TAA) group ( Figure 2B), which were completely (p < 0.0001) repressed by RES ( Figure 2C and D). of rats, MMP-9 mRNA message assessment showed signi cant augmentation in the model group compared with the control group, which was signi cantly (p = 0.0014), but not completely inhibited by RES in the RES +TAA group ( Figure 2E). In addition, a positive correlation (r = 0.858; p < 0.0001) was obtained between α-SMA and MMP-9 ( Figure  2F).

Resveratrol (RES) protects hepatic tissue against TAA-induced brosis and in ammatory cell in ltration
We tested the suggestion that RES can protect liver tissue against damage induced by TAA. Liver tissues obtained from different rat groups were stained with Masson's trichrome and then examined under light microscopy ( Figure 3). Unremarkable ne collagen deposition in the portal area and absence of in ammatory cells in the control rats was observed ( Figure 3A), whereas, liver sections of the untreated TAA group showed signi cant course collagen deposition ( brosis) in the portal area and septum, and in ammatory cells in ltration around the portal tract ( Figure 3B). RES treatment signi cantly (p < 0.0001) inhibited collagen deposition ( Figure 3C and D) to levels comparable to the control group ( Figure 3D).
To support the link between liver brosis induced by TAA we determined the correlation between brosis score and the tissue levels of iNOS, HIF-1α, α-SMA, and MMP-9 Figure 3C Figure 5D), which were signi cantly (p≤0.007) but not completely inhibited by RES. A positive correlation between collagen deposition ( brosis) and HR (r = 0.871; p<0.0001) ( Figure 5E) and MAP (r = 0.804; p<0.0001) ( Figure 5F) was observed.

Discussion
This study investigated the iNOS/HIF-1α/MMP-9/α-SMA axis mediated liver brosis and hypertension by TAA in the presence and absence of RES in an induced chronic liver injury rat model. We used molecular, biochemical, immunohistochemical, and histological approaches to show that TAA intoxication activated liver tissue iNOS/HIF-1α/α-SMA axis associated with the induction of liver brosis and hypertension, and RES was able to block TAA effects. In addition, RES inhibited TAA-induced matrix metalloproteinase-9 (MMP-9). MMP-9 was reported to participate in liver brosis induced by TAA (Lin et al., 2017). Our results proved that the plant phytoalexin, RES, can ameliorate iNOS/HIF-1α/α-SMA axis and liver brosis and hypertension in a TAA-induced chronic liver injury.
The liver is a known target of TAA intoxication that causes liver brosis and cirrhosis (Reif et al., 2004, Al-Hashem et al., 2019, and our data that demonstrated the induction of hepatic brosis, iNOS, HIF-1α, and MMP-9 by TAA (Figures 1-3) are in agreement with the previous report that demonstrated liver brosis induced by cholesterol and the enhancement of iNOS, HIF-1α, MMP-9 in mice that placed iNOS upstream of HIF-1α and MMP-9 (Anavi et al., 2015). They also demonstrated a pro brotic role for iNOS, both in vivo and in vitro. However, HIF-1α gene knockout in macrophages signi cantly reduced iNOS gene expression (Takeda et al., 2010) suggesting that these two signalling molecules modulate each other's expression. Taken together, we demonstrated in this study that TAA intoxication caused a substantial induction of liver iNOS/HIF-1α/α-SMA/MMP-9/collagen mRNA and protein expression and hypertension, which was effectively inhibited by RES for 10 weeks. The authors declare that all data were generated in-house and that no paper mill was used.

Data availability
The data that support the ndings of this study are available on request from the corresponding author.
The data are not publicly available due to con dential handling of our materials because this manuscript data is part of a big project which is underway.

Statements and Declarations
Page 8/16 The authors declare the absence of any con icts of interest.