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. 2021 Mar 11:2021:6632137.
doi: 10.1155/2021/6632137. eCollection 2021.

Pemafibrate Pretreatment Attenuates Apoptosis and Autophagy during Hepatic Ischemia-Reperfusion Injury by Modulating JAK2/STAT3 β/PPAR α Pathway

Ziqi Cheng  1 , Chuanyong Guo  1
Affiliations

Pemafibrate Pretreatment Attenuates Apoptosis and Autophagy during Hepatic Ischemia-Reperfusion Injury by Modulating JAK2/STAT3 β/PPAR α Pathway

Ziqi Cheng et al. PPAR Res. .

Abstract

Hepatic ischemia-reperfusion injury (HIRI) is a common phenomenon in liver transplantation and liver surgery. This article is aimed at clarifying the role of pemafibrate in HIRI through JAK2/STAT3β/PPARα. In the experiment, we divided Balb/c into seven groups, namely, normal control (NC), Sham, PEM (1.0 mg/kg), IRI, IRI + PEM (0.1 mg/kg), IRI + PEM (0.5 mg/kg), and IRI + PEM (1.0 mg/kg). We used biochemical assay, histopathological evaluation, immunohistochemistry, RT-PCR and qRT-PCR, ELISA analysis, and other methods to determine the level of serum AST, ALT, IL-1β, and TNF-α in the liver at three time points (2 h, 8 h, and 24 h) after reperfusion of apoptosis factor, autophagy factor, and the JAK2/STAT3/PPARα content in tissues. Our experiment results showed that the pemafibrate can effectively reduce the level of hepatic IR injury. In addition, pemafibrate has anti-inflammatory, antiapoptotic, and antiautophagy effects, which are mediated by the JAK2/STAT3β/PPARα pathway.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Pemafibrate has no toxic and side effects on liver structure and function. (a) The levels of ALT and AST were shown as mean ± SD by GraphPad Prism 8 (n = 6; P < 0.01). In these three groups, there was no significant difference in transaminase expression between two groups. (b) After H&E staining, the liver was observed under the light microscope (original magnetization, ×200).
Figure 2
Figure 2
Pemafibrate can significantly alleviate the changes of liver structure and function after ischemia-reperfusion. (a) In three time points, the transaminase level of IRI mice increased significantly and improved after treatment, showing a dose-dependent. ALT and AST levels were expressed by mean ± SD. (b) H&E-stained hepatic sections were examined under light microscopy and imaged at a ×20 magnification. In the IRI group, there were a lot of eosinophilic and unorganized necrotic areas (circled by green lines) and increased inflammatory reactions (indicated by black arrows). However, this phenomenon was alleviated by PEM. Data was given as mean ± SD (n = 6, #P < 0.05 for Sham versus IRI, ∗P < 0.05 for IRI + PEM 0.1 mg/kg versus IRI, !P < 0.05 for IRI + PEM 0.1 mg/kg versus IRI + PEM 0.5 mg/kg, and +P < 0.05 for IRI + PEM 0.5 mg/kg versus IRI + PEM 1.0 mg/kg; (a) two-way ANOVA; (b) Student's t-test).
Figure 3
Figure 3
PEM pretreatment inhibits the production of IL-1β, IL-6, and TNF-α in hepatic IRI. (a) The serum IL-1β and TNF-α levels were measured by ELISA and given as mean ± SD at 2, 8, and 24 hours after reperfusion in mice. (b) The relative mRNA levels of IL-1β, IL-6, and TNF-α were evaluated in each group, as shown by qRT-PCR. (c, d) Protein expression of IL-1β, IL-6, and TNF-α was detected by Western blot. (e) Immunohistochemistry was used to detect TNF-α and IL-1β expression in liver tissues (original magnification, ×200). The IOD sum was analyzed with the Image-Pro Plus software 6.0. Data was presented as the mean ± SD (n = 6, #P < 0.05 for Sham versus IRI, ∗P < 0.05 for IRI + PEM 0.1 mg/kg versus IRI, !P < 0.05 for IRI + PEM 0.1 mg/kg versus IRI + PEM 0.5 mg/kg, and +P < 0.05 for IRI + PEM 0.5 mg/kg versus IRI + PEM 1.0 mg/kg; (a, b, d) two-way ANOVA; (e) Student's t-test). Expression levels of IL-1β, TNF-α, and IL-6 were significantly increased in IRI mice. PEM pretreatment dramatically reduced the levels, particularly at 1.0 mg/kg.
Figure 4
Figure 4
PEM pretreatment ameliorates apoptosis and autophagy in hepatic IRI. (a) The relative mRNA levels of Bcl-2, Bax, Caspase 3, Caspase 9, Beclin-1, LC3, and P62. (b, c) Protein expression of apoptosis- and autophagy-related proteins. (d) Immunohistochemistry was used to detect Bcl-2, Bax, Beclin-1, and LC3 expression in liver tissues (original magnification, ×200). The IOD sum of brown area to total area was analyzed with the Image-Pro Plus software 6.0. Data was presented as the mean ± SD (n = 6, #P < 0.05 for Sham versus IRI, ∗P < 0.05 for IRI + PEM 0.1 mg/kg versus IRI, !P < 0.05 for IRI + PEM 0.1 mg/kg versus IRI + PEM 0.5 mg/kg, and +P < 0.05 for IRI + PEM 0.5 mg/kg versus IRI + PEM 1.0 mg/kg; (a, c) two-way ANOVA; (d) Student's t-test). In the IRI group, the expression of P62 and Bcl-2 was significantly lower than that in the NC group, while the others were significantly higher. PEM could reverse this change.
Figure 5
Figure 5
PEM modulates the phosphorylation of JAK2/STAT3β in hepatic IRI. (a, b) Protein expression of PPARα, JAK2, STAT3, and p-STAT3. (c) The quantitative analysis of Western blot results of JAK2/STAT3β/PPARα. Data was presented as the mean ± SD (n = 6, #P < 0.05 for Sham versus IRI, ∗P < 0.05 for IRI + PEM 0.1 mg/kg versus IRI, !P < 0.05 for IRI + PEM 0.1 mg/kg versus IRI + PEM 0.5 mg/kg, and +P < 0.05 for IRI + PEM 0.5 mg/kg versus IRI + PEM 1.0 mg/kg; (b) two-way ANOVA; (c) Student's t-test). The expression of PPARα and p-STAT3β was notably increased, but JAK2 saw a steep drop.
Figure 6
Figure 6
Kupffer cells produce a number of inflammatory mediators, which can bind to cell membrane receptors, promote JAK2, and inhibit p-STAT3β and PPAR alpha. Once PPARα was inhibited, Bcl-2 decreased, and Bax increased, which could stimulate apoptosis and autophagy. Pemafibrate can inhibit the release of inflammatory factors and reduce the subsequent reactions. It could protect the liver from excessive programmed cell death by increasing the levels of p-STAT3β and PPARα in the hepatic IRI.
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