NAFLD, which is a common liver metabolic disease, is a component of metabolic syndrome; it is closely associated with obesity, dyslipidemia, and type 2 diabetes. Although there is currently no specific medicine for NAFLD, dietary interventions and physical activities are still considered as important complementary strategies during treatment for patients with NASH [19]. Here, we investigated whether the application of MK886 had a therapeutic effect in protecting against HFD-induced NAFLD in rats, by alleviating obesity, hepatic steatosis, and inflammation. Our results suggest that MK886 constitutes a promising treatment for NAFLD.
HFD is one of the most common and effective ways to induce NAFLD. Here, after being fed HFD for seven weeks, we established that our rat model exhibited significantly increased serum levels of AST and ALT, in addition to hepatic steatosis; these findings are in agreement with those of previous studies [20–24]. Therefore, we explored the effects of MK886 using this NAFLD model. Surprisingly, after 14 weeks of HFD, serum TG, TC, HDL all decreased, unlike the findings of Cui. et al [24]. (Table 3). We hypothesized that this inconsistency may be caused by the fact that liver function was damaged in the rats, leading to the failure of their normal lipid metabolism. After intervention with MK886, HFD + MK 886 rats gained significantly lower amounts of weight than those in the HFD group, indicating that MK886 intervention significantly reduced body weight, but did not affect the liver weight or liver index (Fig. 2 and Table 2). Furthermore, HFD + MK886 rats exhibited significantly lower serum levels of AST, ALT, TG, TC, and LDL-C (Table 2 and Table 3). The oil red O results suggest that the amelioration of hepatic steatosis occurred (Fig. 3). In addition, there was excess lipid accumulation in the body and organs, which is considered to be the primary indicator for the pathogenesis of NAFLD, followed by the inducement of inflammatory response and oxidative stress [25]. Previous studies have confirmed that IL-6, TNF-α, and MCP-1 are the most common cytokines involved in the inflammatory response [4, 25]. Here, after 14 weeks of HFD, we observed that levels of IL-6, TNF-α, and MCP-1 increased. After the application of MK886, inflammatory parameters significantly decreased (Table 4), while typical histological features including steatosis, inflammation, ballooning degeneration, and NAS showed downward trends (Fig. 4). Above all, these results demonstrate that MK886 ameliorates lipid metabolism and improves liver function and systemic inflammation in rats. To further explore the mechanism of FA metabolism, we detected protein expressions through western blot analysis.
PPARα was the first member of the nuclear receptor superfamily to be identified; it is mainly expressed in tissues with high-energy metabolisms, such as liver, kidney, heart, and muscle tissues [8]. Regarding the pathogenesis of NAFLD, PPARα generally plays a crucial role in controlling FA β-oxidation; thus, it is considered to be a promising target for the treatment of NAFLD, due to its excellent activity in maintaining lipid homeostasis [10]. Here, we found that the level of PPARα was significantly lower in the HFD group than it in the CON group. We hypothesize that HFD increases lipid accumulation by decreasing PPARα expression. Furthermore, MK886 significantly decreased its levels. During transcription, PPARα activates several genes, including those encoding CD36 and FABPs [26]. Previous studies have suggested that inhibiting FFA uptake by targeting CD36 transcripts [27] and inhibiting the expression of FABP1 [28] constitutes a promising therapeutic strategy for treating NAFLD. CPT1A is considered a rate-limiting enzyme of β-oxidation, regulated by PPARα [29]. In contrast to adipose tissue, PPARγ expression is lower in the liver. However, under a pathophysiologic situation, such as the overload of dietary lipids, hepatic PPARγ expression can be induced [30]. As shown by the protein bands in Fig. 5A, we found that the expression of CPT1A (Fig. 5C) decreased significantly in the HFD group compared with the CON group, while the expression of CD36 (Fig. 5E) increased significantly; PPARγ (Fig. 5D) and FABP1 (Fig. 5F) showed insignificant changes. After the application of MK886, levels of CPT1A (Fig. 5C), PPARγ (Fig. 5D), and CD36 (Fig. 5E) decreased significantly, but FABP1 (Fig. 5F) did not change significantly. Thus, HFD appeared to reduce the β-oxidation of FA by inhibiting PPARα and CPT1A, while it upregulated the expression of CD36 to increase FA uptake, ultimately leading to liver steatosis. MK886 significantly reduced the expressions of PPARα, CPT1A, PPARγ, and CD36, and inhibited fatty acid β-oxidation; however, it reduced the intake of FA.
MK886 was originally identified as an inhibitor of FLAP, and this protein is an integral part of 5-lipoxygenase (5-LOX). It is not only a fatty-acid binding protein but also plays an important role in apoptosis in other tissues [31, 32]. In this study, we mainly discussed the lipid metabolism function of MK886, as an antagonist of PPARα, in NAFLD, so we mainly selected liver tissues to measure the expression of related proteins. We discovered that MK886 can improve hepatic function, systematic inflammation, and hepatic steatosis, and can also potentially influence liver injury. Therefore, MK886 has the potential to improve hepatic lipid metabolism. Surprisingly, we discovered that MK886 antagonizes both PPARα and PPARγ, similar to the findings of Zhao X. et al [33]. We hypothesize that PPARα and PPARγ might have combined meditated effects, as predicted by Jain. et al [34]. Therefore, MK886 might be a dual PPARα/PPARγ antagonist. However, this study has some limitations. First, recent developments have demonstrated the complexities of mechanisms including lipid metabolism, inflammatory response, oxidative stress, and the gut microbiome [1, 35]. Therefore, it is impossible to ameliorate disease from only one single pathway. Besides, we mainly explored protein expression during the occurrence and development of NAFLD without consideration of related gene levels. Second, here we only explored the effects of MK886 on lipid uptake and β-oxidation; its effects on DNL and lipid export should also be explored by future studies. Thirdly, perhaps the rat model is not the most appropriate for NAFLD, the characteristics of the disease are not constant as the disease developed compared with the mouse model. But considering the periodicity of drug stimulation and the visual change on energy intake and body mass, we chose rat model with HFD, as in previous studies [36, 37]. In addition, clinical trials are required to build upon the fundamental findings of animal studies