**3. The role of hypertension in the progression of NASH**

SHRSP5/Dmcr rats are hypertensive, making this strain an ideal model in which to study the correlation between hypertension and NASH. In our previous study, we investigated the mechanism underlying the development of hypertension-associated NASH using three strains of a rat: normotensive WKY, hypertensive SHR and SHRSP5/Dmcr [28]. As mentioned previously, SHRSP5/Dmcr was established from the SHRSP strain, which was derived from SHR strain that was developed from normotensive WKY rats by selective inbreeding of the rats with spontaneously high systolic blood pressure in normal conditions [29]. Male rats with a blood pressure of 150–175 mmHg persisting for more than 1 month, and females with a blood pressure of 130– 140 mmHg were mated, and the offspring with high blood pressure (over 150 mmHg persisting for more than 1 month) were selected for further inbreeding. The severity of hypertension was elevated from generation to generation, and all the rats from the third to sixth generation developed spontaneous hypertension by 15 weeks of age. Since the SHR and WKY originated from the same parental outbred Wistar rats, the WKY strain was used as the normotensive control for the SHR and SHRSP5/Dmcr strains. The blood pressure in the adult male rats of the three strains, WKY, SHR, and SHRSP5/Dmcr, were 130, 235, and 180 mmHg, respectively [28].

In our study, the normotensive WKY strain, and two hypertensive SHR and SHRSP5/Dmcr strains were fed either the HFC or control diet for 8 weeks. Changes to liver pathology and expression of proteins associated with inflammation and oxidative stress were determined [28]. We evaluated serum levels of AST, ALT, and γ-GTP, and confirmed that mild liver damage occurred in the hypertensive strains in the absence of HFC feeding, suggesting that hypertension may be a risk factor for chronic liver disease. The HFC diet induced more severe lobular inflammation and hepatic fibrosis in the hypertensive strains compared with the normotensive strain. The severity of the hepatic fibrosis observed in the SHRSP5/Dmcr strain was even higher compared with that of the SHR strain. The HFC diet induced elevation of serum inflammatory cytokines, TNF-α and TGF-β1, in the hypertensive strains, whereas an increase in TGF-β1 was not observed in the normotensive rats. The combination of TNF-α and TGF-β1 may trigger a more severe inflammatory response in the hypertensive rats by regulating the activation of downstream inflammatory signaling such as NF-κB and mitogen-activated protein kinase (MAPK) pathways. Increased activation of NF-κB and MAPK (p38 and JNK) signaling occurred in the hypertensive strains, which may have contributed to the more severe lobular inflammation observed in these rats. In addition, oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and their elimination by antioxidant defenses, may lead to cellular injury and chronic inflammation [30]. An increase in oxidative stress in NASH patients was previously reported [31, 32]. We measured serum thiobarbituric acid reactive substances levels and found that oxidative stress was significantly elevated in hypertensive strains fed an HFC diet but not in normotensive rats (unpublished data). Meanwhile, in hypertensive rats, the HFC diet suppressed the nuclear factor erythroid 2-related factor 2 (Nrf2)/ Kelch-like ECH-associated protein 1 (Keap1) pathway, involved in antioxidative defenses [33]. We also found that hepatic levels of superoxide dismutase-1 (SOD-1) [25] and SOD-2 [28], that contribute to antioxidant defense by catalyzing the dismutation of superoxide anions [34], were decreased in hypertensive SHRSP5/Dmcr rats fed the HFC diet. The decrease in SOD-2 expression induced by HFC feeding was not observed in normotensive WKY and hypertensive SHR strains [28]. This could suggest that an increase in oxidative stress and a lower antioxidative capacity may trigger a more severe inflammatory response and liver damage in hypertensive rat strains following HFC feeding, compared with normotensive strains.

cell (HSC) activation, which results in the production and deposition of extracellular matrix (ECM) [27]. The HFC diet induced the upregulation of transforming growth factor-β1 (TGFβ1), a profibrotic cytokine that promotes HSC activation, prior to the appearance of obvious hepatic fibrosis (at 2 weeks). Its upregulation was also observed at subsequent stages (at 8 and 16 weeks). Expression of alpha smooth muscle actin (α-SMA) and platelet-derived growth factor-B, involved in hepatic fibrosis, were elevated at 8 weeks of HFC feeding, indicating extensive activation of HSC at this time point. Alpha-1 type I collagen, the major component of ECM, was produced by activated HSC (myofibroblast) and was markedly elevated at 8 and 16 weeks, corresponding to the appearance of extensive liver fibrosis observed at the same time points.

In order to investigate the role of dietary cholesterol in the pathogenesis of HFC diet-induced NASH in SHRSP5/Dmcr rats, we compared hepatic histological changes induced by a high fat (HF) diet and those by an HFC diet (unpublished). As described above, the HFC diet induced severe steatosis, lymphocyte infiltration, ballooned hepatocytes, and fibrosis in the livers of the rats. In contrast, HF feeding only led to mild hepatic steatosis and lymphocyte infiltration, while liver fibrosis was not observed. It was suggested that dietary cholesterol may play a key role in the transition from simple steatosis to fibrotic steatohepatitis, the progressive stage,

SHRSP5/Dmcr rats are hypertensive, making this strain an ideal model in which to study the correlation between hypertension and NASH. In our previous study, we investigated the mechanism underlying the development of hypertension-associated NASH using three strains of a rat: normotensive WKY, hypertensive SHR and SHRSP5/Dmcr [28]. As mentioned previously, SHRSP5/Dmcr was established from the SHRSP strain, which was derived from SHR strain that was developed from normotensive WKY rats by selective inbreeding of the rats with spontaneously high systolic blood pressure in normal conditions [29]. Male rats with a blood pressure of 150–175 mmHg persisting for more than 1 month, and females with a blood pressure of 130– 140 mmHg were mated, and the offspring with high blood pressure (over 150 mmHg persisting for more than 1 month) were selected for further inbreeding. The severity of hypertension was elevated from generation to generation, and all the rats from the third to sixth generation developed spontaneous hypertension by 15 weeks of age. Since the SHR and WKY originated from the same parental outbred Wistar rats, the WKY strain was used as the normotensive control for the SHR and SHRSP5/Dmcr strains. The blood pressure in the adult male rats of the three strains, WKY, SHR, and SHRSP5/Dmcr, were 130, 235, and 180 mmHg, respectively [28].

In our study, the normotensive WKY strain, and two hypertensive SHR and SHRSP5/Dmcr strains were fed either the HFC or control diet for 8 weeks. Changes to liver pathology and expression of proteins associated with inflammation and oxidative stress were determined [28]. We evaluated serum levels of AST, ALT, and γ-GTP, and confirmed that mild liver damage occurred in the hypertensive strains in the absence of HFC feeding, suggesting that hypertension may be a risk factor for chronic liver disease. The HFC diet induced more severe lobular inflammation and hepatic fibrosis in the hypertensive strains compared with the normotensive

during the progression of NAFLD/NASH.

112 Cholesterol - Good, Bad and the Heart

**3. The role of hypertension in the progression of NASH**
