Studies On The Role Of PPARα/γ Signaling Pathway In The Pathogenesis Of Hyperlipidemic Steatohepatitis

Objective:To study the role of nuclear receptor PPARα/γ signaling pathway in the pathogenesis of hyperlipidemic steatohepatitis, and provide the basis for the rational use of acting on PPARα/γ target drugs for hyperlipidemic steatohepatitic treatment.Methods: Male SD rats were used and randomly divided into eight groups: control group, model group, PPARα agonist(fenofibrate 20 mg/kg) group, PPARγ agonist(rosiglitazone 4 mg/kg) group, fenofibrate 20 mg/kg plus rosiglitazone 4 mg/kg group, PPARα antagonist(MK886 1 mg/kg) group, PPARγ antagonist(GW9662 1 mg/kg) group, and MK886 1 mg/kg plus GW9662 1 mg/kg group. The rat model was established by orally feeding high-fat and high-sucrose emulsion by gavage when they were simultaneously treated with drugs for 6 weeks. Thereafter, the liver and blood were taken, the total cholesterol(TC), triglycerides(TG), free fatty acids(FFA), superoxide dismutase(SOD), malondialdehyde(MDA), glutathione(GSH), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), IL-8, and monocyte chemoattractant protein 1(MCP-1) in serum and hepatic tissue, alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in serum, and liver weight were measured, and coefficient of hepatic weight were calculated. The histological changes of liver specimens were also examined. The m RNA and protein expressions of hepatic peroxisome proliferator-activated receptor(PPAR) α/γ, sterol regulatory element binding protein(SREBP)-1c, fatty acid synthase(FAS), diacylglycerol acyltransferase(DGAT), lipoprotein lipase(LPL), and protein expressions of hepatic nuclear factor kappa B(NF-κB) and inhibitor-K binding protein α(IκBα) were examined by RT-PCR and Western blot methods, respectively.The LPS and oleic acid- stimulated BRL cells were used to observe the effects of PPARα/γ agonists(fenofibrate and/or rosiglitazone) and antagonists(MK886 and/or GW9662) on cultured supernatant IL-6, TNF-α, ALT, AST, and MDA contents as well as intracellular PPARα/γ, SREBP-1c, FAS, DGAT, CPT1 A, LPL, NF-κB, and IκB-α protein expressions. At the same time, the nuclear translocation of NF-κB was determined by immunocytochemistry method.Results: After simultaneous administration of PPARα/γ agonist(fenofibrate and/or rosiglitazone) and PPARα/γ antagonist(MK886 and/or GW9662) for 6 weeks, the serum and hepatic tissue levels of TC and TG were significantly decreased and increased(P<0.05 or P<0.01), especially PPARα/γ agonists or PPARα/γ antagonists co-treatment. Fenofibrate could also increase and decrease the levels of serum and hepatic tissue FFA(P<0.01) respectively, while rosiglitazone could reduce the serum and hepatic tissue FFA(P<0.01); PPARα/γ agonists co-administration could reduce the level of hepatic tissue FFA(P<0.01), but the obvious change of serum FFA level was not observed. After administration of PPARα antagonist, the serum level of FFA significantly decreased(P<0.01), whereas PPARγ antagonist, PPARα/γ antagonist co-administration could significantly increase the serum FFA level(P<0.01), but PPARα antagonist or/and PPARγ antagonist administration did not change hepatic FFA level. These results indicated that after administration of fenofibrate or rosiglitazone, the serum level of FFA was obviously different, but the hepatic tissue level of FFA was decreased. Therefore, co-PPARα/γ agonists might exert the beneficial effect on lipid metabolism. PPARα/γ agonists or antagonists use alone and in combination could significantly increase the levels of hepatic GSH, SOD, and serum GSH contents(P<0.05 or P<0.01), and PPARα/γ agonists alone and in combination use could significantly increase the serum SOD content(P<0.01). PPARα/γ agonists alone and in conjunction could significantly decrease the serum and hepatic MDA level(P<0.05 or P<0.01), conversely, PPARα/γ antagonist could significantly increase the serum MDA level(P<0.01), but the hepatic MDA content was not affected. The results suggested that co-PPARα/γ agonists administration could significantly decrease the serum and hepatic MDA content, and increase the hepatic GSH content, which might play a beneficial antioxidant effect. PPARα/γ agonists could significantly reduce serum and hepatic TNF-α, IL-6, IL-8, and MCP-1 levels(P<0.05 or P<0.01), on the contrary, PPARα/γ antagonist could increase these inflammatory cytokines in some degrees, especially in MK886 plus GW9662-treated group(P<0.05 or P<0.01). Meanwhile, rosiglitazone could significantly reduce the serum ALT level(P<0.01). However, in the fenofibrate, fenofibrate plus rosiglitazone, and PPARα/γ antagonist groups, the serum levels of ALT and AST did not changed. In contrast to the PPARα agonist, PPARγ agonist treatment might decrease the hepatic MDA content and serum ALT level, in parallel, the serum and hepatic inflammatory cytokines were also reduced, which suggested that the hepatic oxidative stress and inflammatory cytokines might mainly be controlled by PPARγ. Additionally, fenofibrate and fenofibrate plus rosiglitazone treatments could significantly increase liver weight and coefficient of liver weight(P<0.01), whereas rosiglitazone could reduce the both indexes(P<0.05 or P<0.01), but fenofibrate plus/or rosiglitazone treatments had no significant effect on body weight. When simultaneous administration of PPARα/γ antagonists, the body weight, liver weight, and coefficients of liver weight also had no significant changes. Importantly, fenofibrate or fenofibrate plus rosiglitazone treatment might significantly reduced the fatty degeneration of liver(P<0.01); the inflammatory cell infiltration also significantly reduced in the fenofibrate and rosiglitazone alone and co-administration-treated groups(P<0.05), the necrosis of the liver was significantly improved as well after administration of rosiglitazone and fenofibrate plus rosiglitazone(P<0.05). Fenofibrate and rosiglitazone used alone and co-administration could significantly increase the hepatic PPARα, PPARγ, LPL m RNA and protein expressions as well as IκB-α protein expression, and reduce the hepatic SREBP-1c, FAS, DGAT m RNA and protein expression as well as NF-κB protein expression(P<0.01). On the contrary, after administration of MK886 or MK886 plus GW9662, PPARα m RNA and protein expressions were significantly decrease(P<0.01); while GW9662 or GW9662 plus MK886 treatment could significantly decrease the PPARγ m RNA and protein expressions(P<0.01). MK886 and GW966 used alone and co-administration could significantly reduce the hepatic LPL m RNA and protein expressions and IκB-α protein expression, but hepatic SREBP-1c, FAS, and DGAT m RNA and protein expressions as well as NF-κB protein expression were significantly increased(P<0.01). After administration of PPARα/γ agonist or PPARα/γ antagonist,the lipid metabolism and inflammation gene expressions were in accordance with the serum and hepatic lipid and inflammatory cytokine levels, which suggested that PPARα/γ play an important role in the pathogenesis of hyperlipidemic steatohepatitis.In vitro, after pretreatments with PPARα/γ agonists(fenofibrate and/or rosiglitazone) and PPARγ antagonist GW9662, fenofibrate plus GW9662 for 2h, PPARα protein expression in oleic acid(200 μmol/L) 36 h plus LPS(1μg/m L) 9 h-stimulated BRL cells was significantly upregulated(P<0.01); whereas pretreatments with MK886, MK886 plus rosiglitazone, the PPARα protein expression was significantly inhibited(P<0.05 or P<0.01). Likewise, after pretreatments with fenofibrate or/and rosiglitazone, MK886, MK886 plus rosiglitazone, PPARγ protein expression was significantly upregulated(P<0.05 or P<0.01); whereas pretreatments with GW9662, fenofibrate plus GW9662, PPARγ protein expression was inhibited(P<0.05 or P<0.01). Following pretreatments with fenofibrate or/and rosiglitazone, MK886 plus pioglitazone, and fenofibrate plus GW9662, the SREBP-1c, FAS, and DGAT protein expressions were obviously down-regulated, while the CPT1 A and LPL protein expressions were obviously up-regulated, and the reverse results were observed in the MK886- and GW9662-treated groups(P<0.05 or P<0.01). In addition, oil red O staining found that, after pretreatments with PPARα/γ agonists and PPARα/γ antagonists could respectively decrease and increase the lipid droplets in oleic acid-stimulated BRL cells. The level of MDA was significantly reduced after fenofibrate or/and rosiglitazone, MK886 plus rosiglitazone, and fenofibrate plus GW9662 pretreatments, and significantly increased after MK886 and GW9662 pretreatments(P<0.05 or P<0.01). Fenofibrate or/and rosiglitazone pretreatments could decrease ALT and AST levels, but MK886 and GW9662 pretreatments could significantly increase ALT and AST levels(P<0.05 or P<0.01). Additionally, pretreatments with fenofibrate or/and rosiglitazone, MK886 plus rosiglitazone, and fenofibrate plus GW9662, could significantly reduce TNF-α and IL-6 levels, reversely, MK886 and GW9662 pretreatments could significantly increase the both cytokine level(P<0.05 or P<0.01). Addition of fenofibrate or/and rosiglitazone, and MK886 plus rosiglitazone pretreatments might significantly down-regulate NF-κB and up-regulate IκB-α protein expressions, and reduce the NF-κB nuclear translocation, respectively; fenofibrate and GW9662 pretreatments might also reduce NF-κB nuclear translocation, but the reverse results were observed after addition of MK886 and GW9662 pretreatments(P<0.01). These results suggested that, after administration of PPARα/γ agonist or PPARα/γ antagonist,the lipid metabolism, oxidative stress, and inflammation cytokines were in accordance with the in vivo results.Conclusion: PPARα/γ signaling pathway involved in the pathogenesis of hyperlipidemic steatohepatitic formation by regulating lipid metabolism, oxidative stree, and inflammatory cytokine production. The results demonstrated that PPARα agonist plus PPARγ antagonist or PPARγ agonist plus PPARα antagonist might improve the abnormal pathological changes in cultured cells in vitro, and the more effective efficacy were observed in the co-PPARα/γ agonist-treated group in vivo and in vitro. These findings suggested that PPARα/γ might be the potential targets for hyperlipidemic steatohepatitic treatment.