Jun-Ling Pu ,Zuo-Tin Hung ,Yun-Hi Luo ,Tong Mou ,Ting-Ting Li ,Zhong-Tng Li ,Xu-Fu Wei,Zhong-Jun Wu,*

a Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 40 0 016, China

b Department of Anatomy, Basic Medical College, Chongqing Medical University, Chongqing 40 0 016, China

Keywords:Fisetin Hepatic ischemia-reperfusion injury GSK3 β AMPK NLRP3

ABSTRACT Background: Hepatic ischemia-reperfusion (I/R) injury (IRI) represents a crucial challenge in liver transplantation.Fisetin has anti-inflammatory,anti-aging and anti-oxidative properties.This study aimed to examine whether fisetin mitigates hepatic IRI and examine its underlying mechanisms.Methods: Sham or warm hepatic I/R operated mice were pretreated with fisetin (5,10 or 20 mg/kg).Hepatic histological assessments,TUNEL assays and serum aminotransferase measurements were performed.An in vitro hypoxia/reoxygenation (H/R) model using RAW264.7 macrophages pretreated with fisetin (2.5,5 or 10 μmol/L) was also used.Serum and cell supernatant concentrations of interleukin-1 β (IL-1 β),IL-18 and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA).Protein levels of p-GSK3 β,p-AMPK and NLR family pyrin domain-containing 3 (NLRP3)-associated proteins were detected by Western blotting.Results: Compared with the I/R group,fisetin pretreatment reduced pathological liver damage,serum aminotransferase levels,serum concentrations of IL-1 β,IL-18 and TNF-α in the murine IRI model.Fisetin also reduced the expression of NLRP3 inflammasome-associated proteins (NLRP3,cleaved caspase-1,IL-1 β and IL-18) in I/R-operated liver.The experiments in vitro showed that fisetin decreased the release of IL-1 β,IL-18 and TNF-α,and reduced the expression of NLRP3 inflammasome-associated proteins in H/R-treated RAW264.7 cells.Moreover,fisetin increased the expressions of p-GSK3 β and p-AMPK in both models,indicating that its anti-inflammatory effects were dependent on GSK3 β/AMPK signaling.The antiinflammatory effects of fisetin were partially inhibited by the AMPK specific inhibitor compound C.Conclusions: Fisetin showed protective effects against hepatic IRI,countering inflammatory responses through mediating the GSK3 β/AMPK/NLRP3 inflammasome pathway.

Introduction

Hepatic ischemia-reperfusion (I/R) injury (IRI) represents a crucial challenge for patients undergoing liver surgical procedures,especially for liver transplants [1] .Increased ischemia time and exacerbated hepatic IRI may cause postoperative complications including early graft dysfunction and graft rejection,threatening the survival of patients [2,3].The underlying physiopathology of I/R damage is complex,and excessive inflammatory response was considered a critical process during I/R [4,5].As an essential tissue-damage detector,the NLR family pyrin domain-containing 3(NLRP3) inflammasome is prominent for initiating and amplifying the inflammatory response.Moreover,accumulating evidence implicates the involvement of NLRP3 inflammasome in the pathogenesis of hepatic IRI [6-8].

The NLRP3 inflammasome is activated when cellular homeostasis is disturbed,either through endogenous (tissue injury)or pathogenic (infection) signals,followed by caspase-1 autoactivation.Thereafter,caspase-1 processes precursor interleukin-1β(proIL-1β) and proIL-18 into active forms,followed with their release [9,10].With respect to relieving hepatic I/R damage,potential therapeutic targets have been identified,including the glycogen synthase kinase 3β(GSK3β) [11] .GSK3βregulates various physiological processes including metabolism,apoptosis,and immunoregulation [12] .As a constitutively active protein kinase,the enzymatic activity of GSK3βis inhibited by phosphorylation at Ser9 and enhanced by phosphorylation at Tyr216.It is noteworthy that GSK3βinhibition through Ser9 phosphorylation has been demonstrated to successfully relieve hepatic IRI [11] .In addition,GSK3βalso regulates the activity of adenosine monophosphateactivated protein kinase (AMPK),a well-known metabolic regulator [13] .AMPK was shown to alleviate hepatic IRI through inhibiting macrophage activation [14] .The recent evidence suggests that the NLRP3 inflammasome might be regulated by AMPK in macrophages [15] .Thus,this collective evidence proposes that NLRP3 inflammasome regulation occurs through the GSK3β/AMPK pathway.

Fisetin is a plant bioflavonoid extracted from fruits,including apples,strawberries and persimmons.It was shown to have therapeutic potential in cancer,inflammatory and metabolic diseases [16] .Furthermore,several studies indicated that fisetin could mitigate myocardial IRI [17,18].Interestingly,fisetin was documented to have a regulatory effect on GSK3βand AMPK [17,19].The present study was to define the potential of fisetin to alleviate hepatic IRI,particularly its actions against GSK3β/AMPK/NLRP3 inflammasome signaling.

Materials and methods

Preparation of fisetin

Fisetin (purity＞98%) provided by MedChemExpress (Monmouth Junction,NJ,USA) was dissolved by DMSO (Sigma,St.Louis,MO,USA) and further diluted by saline or cell culture medium before use.

Animals

Male C57BL/6J mice (weighing 25-28 g) were supplied by the Experimental Animal Center of Chongqing Medical University (Chongqing,China).The animals were bred under specific pathogen-free conditions.All animal experiments were authorized by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (2020-514).

Surgical procedures

Thirty C57BL/6J mice were randomized into six groups (n=5 each group):sham group,sham+FIS-20 group (sham with 20 mg/kg fisetin pretreatment),I/R group (I/R operation alone),and I/R with three concentrations of fisetin pretreatment groups (5,10 or 20 mg/kg,respectively).Mice were anesthetized with pentobarbital (40 mg/kg,Merck,Kenilworth,NJ,USA) by intraperitoneal injection.For I/R operation groups,a 70% warm hepatic I/R surgery was performed as described [20] .Briefly,the portal vein and hepatic artery were occluded by arterial clips for 1 h and then reperfused for 6 h.The sham group underwent similar procedures without clamping.For fisetin pretreatment groups,fisetin was administered intraperitoneally 0.5 h before operation.All mice were sacrificed after 6 h upon reperfusion.

Macrophage culture

RAW264.7 macrophages were provided by Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences and cultured in Dulbecco’s Modified Eagle’s Medium (Gibco,Grand Island,NY,USA) containing 10% fetal bovine serum (HyClone,Logan,Utah,USA) at 37 °C and 5% CO2.

Hypoxia/reoxygenation (H/R) in vitro model

RAW264.7 macrophages were exposed to hypoxic culture conditions for 6 h (1% O2and 5% CO2) in a triple gas incubator(Thermo,Waltham,MA,USA) before storing cells to normoxia for 6 h as previously described [20] .Cells were treated with 2.5,5 or 10 μmol/L fisetin during H/R along with the AMPK inhibitor compound C (Comp.C;MedChemExpress) with the following experimental groups used:control group,FIS-10 group (10 μmol/L fisetin),H/R group,H/R+FIS-2.5 group (H/R with 2.5 μmol/L fisetin),H/R+FIS-5 group (5 μmol/L fisetin),H/R+FIS-10 group(10 μmol/L fisetin),H/R+Comp.C group (10 μmol/L compound C) and H/R+FIS-10+Comp.C group (10 μmol/L fisetin and compound C,respectively).Cell lysates and supernatants were obtained for further analysis.

Hematoxylin and eosin (HE) staining and TUNEL assays

Paraformaldehyde-fixed paraffin-embedded liver tissue specimens were sectioned and subjected to standard HE staining.Afterwards,the severity of hepatic pathological impairment was evaluated by the Suzuki’s injury scoring system [21] .For assessment of apoptosis,sections were stained using the TUNEL kit (Roche,Basel,Switzerland) following the manufacturer’s illustrations.

Aminotransferase assay

Serum samples were used to analyze the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)using special aminotransferase assay kits (Nanjing Jiancheng Bioengineering Institute,Nanjing,China) following the manufacturer’s instructions.

Cytokines determination

IL-1β,IL-18 and tumor necrosis factor-α(TNF-α) concentrations in serum and cell supernatants were determined by enzyme-linked immunosorbent assay (ELISA) kits (Neobioscience,Beijing,China)following the manufacturer’s instructions.

Western blotting

Tissue and cell lysates were prepared using the RIPA kit(Boster,Wuhan,China) [22] .Equal amounts of soluble protein(measured by BCA method) were resolved by SDS-PAGE,transferred to PVDF membranes before analysis by Western blotting using antibodies directed against p-GSK3β(Ser9,Bioss,Beijing,China),GSK3β(Proteintech,Wuhan,China),p-AMPK (Thr172,ABclonal,Wuhan,China),AMPK (Proteintech),NLRP3 (Bioss),IL-1β(Wanleibio,Shanghai,China),proIL-1β(Wanleibio),IL-18 (Proteintech),proIL-18 (Bioss),cleaved caspase-1 (Abcam,Cambridge,UK),caspase-1 (Abcam) andβ-actin (Proteintech).

Statistical analysis

Data analyses were performed using GraphPad Prism v8.0(GraphPad Software,San Diego,CA,USA).All data were expressed as mean ± standard deviation (SD).One-way analysis of variance(ANOVA) tests with Turkey’sposthoctests were used to identify statistical differences among multiple groups.APvalue＜0.05(two-tailed) was defined as statistically significant.

Fig.1.Fisetin improves liver function and decreases inflammatory cytokines after I/R operation in mice.n=5/group.** P ＜ 0.01,vs.Sham;## P ＜ 0.01,vs.I/R.

Results

Fisetin improves liver function and decreases inflammatory cytokines after I / R operation

As the primary diagnostic parameters of liver function,serum ALT and AST concentrations were significantly elevated after the 70% warm hepatic I/R operation.Notably,pretreatment with increasing doses of fisetin (5,10 or 20 mg/kg) gradually reduced ALT and AST concentrations compared to those of the I/R group (Fig.1 A and B,P＜0.01),suggesting that fisetin alleviated hepatic IRI.

As shown in Fig.1 C-E,we observed significantly increases in IL-1β,IL-18 and TNF-αafter I/R surgery (P＜0.01).However,fisetin pretreatment (5,10 or 20 mg/kg) gradually attenuated the increased concentrations of these cytokines compared to those of the I/R group (P＜0.01).

Fisetin decreases the severity of histopathological changes in the I/R model

Examination of liver sections from the sham group revealed liver lobule structures arranged in an orderly manner,the hepatic cords were clear and there was almost no hepatic injury or necrosis (Fig.2 A).Similar findings were observed in the sham+FIS-20 group,indicating that fisetin had no impact on the normal liver condition.In contrast,representative pathological changes including coagulation necrosis,severe architectural abnormalities,sinusoid congestion and vacuolization were found after I/R operation.Instructively,fisetin pretreatment (5,10 or 20 mg/kg) significantly reduced these hepatic pathological changes.These findings were confirmed after assessing liver sections using Suzuki’s scores(Fig.2 C).

Fisetin attenuates hepatocyte apoptosis

Consistent with the results of HE staining,the sham and the sham+FIS-20 groups had few TUNEL positive cells which dramatically increased after I/R operation (Fig.2 B).However,compared to the I/R group,fisetin pretreatment (5,10 or 20 mg/kg) reduced the number of TUNEL positive cells in a dose-dependent manner(Fig.2 B and D,P＜0.01).

Fisetin regulates the GSK3 β/AMPK/NLRP3 inflammasome pathway in the I / R model

After the I/R procedure,liver tissues exhibited significant increase in the expression of NLRP3,cleaved caspase-1,IL-1βand IL-18 compared to those of the sham group,while p-GSK3β(Ser9)and p-AMPK (Thr172) protein expressions were decreased (Fig.3,P＜0.01).However,fisetin pretreatment (5,10 or 20 mg/kg) gradually diminished the activation of NLRP3 inflammasome-associated proteins along with the elevated levels of p-GSK3β(Ser9) and p-AMPK (Thr172) (Fig.3,P＜0.05).

Fisetin mitigates H/R-induced inflammation via the GSK3 β/AMPK/NLRP3 inflammasome pathway in vitro

Notably,we observed significantly elevated IL-1β,IL-18 and TNF-αproduction in cell supernatants after exposure to H/R conditions (Fig.4 A-C,P＜0.01).Remarkably,fisetin treatment (2.5,5 or 10 μmol/L) gradually diminished the concentrations of these cytokines (Fig.4 A-C,P＜0.05).

Indeed,H/R exposure resulted in significant elevations in the protein levels of NLRP3,cleaved caspase-1,IL-1βand IL-18,while the expressions of p-GSK3β(Ser9) and p-AMPK (Thr172) were diminished (Fig.4 D-J,P＜0.01).Treatment with fisetin (2.5,5 or 10 μmol/L) gradually diminished activation of the NLRP3 inflammasome as indicated by inhibition of the increased levels of NLRP3,cleaved caspase-1,IL-1βand IL-18,and enhancement of p-GSK3β(Ser9) and p-AMPK (Thr172) expression (Fig.4 D,P＜0.05).

Fig.2.Fisetin attenuates hepatic histopathological changes and hepatocyte apoptosis after I/R operation in mice.A and C:Representative HE liver sections (original magnification × 200) and Suzuki’s score results of the different treatment groups;B and D:representative TUNEL staining of liver sections (original magnification × 400) and estimation of TUNEL positive cell numbers.n=5/group.** P ＜ 0.01,vs.Sham;## P ＜ 0.01,vs.I/R.

Fig.3.Fisetin regulates GSK3 β/AMPK/NLRP3 inflammasome proteins in the liver I/R model.A:Representative Western blotting of p-GSK3 β (Ser9),p-AMPK (Thr172) and NLRP3 inflammasome-associated proteins in mouse liver tissues among the treatment groups.B-G:Quantitative analysis of all samples from A .n=5/group.** P ＜ 0.01,vs.Sham;# P ＜ 0.05,## P ＜ 0.01,vs.I/R.

Protective effects of fisetin are attenuated after inhibiting AMPK

Compared to the H/R+FIS-10 (H/R+10 μmol/L fisetin) group,compound C significantly elevated the inflammatory cytokines(Fig.5 A-C,P＜0.01).Moreover,Western blotting analysis indicated that compared with the H/R+FIS-10 group,compound C treatment (H/R+FIS-10+Comp.C group) significantly boosted the expression of NLRP3,cleaved caspase-1,IL-1βand IL-18,and reduced the p-AMPK (Thr172) expression (Fig.5 D-J,P＜0.05).However,compound C treatment did not entirely block the regulation effect of fisetin on NLRP3 inflammasome.Interestingly,there was no significant difference in the expression of p-GSK3β(Ser9) between the two groups (Fig.5 D and E,P＞0.05),suggesting that GSK3β(Ser9) phosphorylation was not regulated by AMPK in this scenario.

Discussion

As an inevitable obstacle in liver transplant operations,hepatic IRI has attracted considerable attention.As is commonly known,a longer ischemia time implies a higher risk of postoperative complications and worse prognosis [23] .Our previous studies indicated that inhibiting inflammatory reactions by targeting proinflammatory molecules may mitigate hepatic IRI [20,24].Thus,pharmacological interventions targeting specific molecules may contribute to the development of hepatic IRI treatments.Herein,we verified that fisetin improved liver function,decreased inflammatory cytokines and attenuated hepatic histopathological changesinvivo,as well as suppressing inflammatory actionsinvitro.Moreover,we link the protective effects of fisetin with regulation of the GSK3β/AMPK/NLRP3 inflammasome pathway.Our findings may provide some support for the clinical treatment of hepatic IRI.

Fig.4.Fisetin inhibits inflammation in H/R-exposed macrophages by regulating the GSK3 β/AMPK/NLRP3 inflammasome pathway.A-C:Production of IL-1 β,IL-18 and TNF-α in RAW264.7 macrophages was measured by ELISA in the indicated treatment groups;D-J:relative expression of p-GSK3 β (Ser9),p-AMPK (Thr172) and NLRP3 inflammasome-associated proteins in the cells from A .** P ＜ 0.01,vs.Control;# P ＜ 0.05,## P ＜ 0.01,vs.H/R.

The most salient finding of our study involved the proved benefit of fisetin in relieving hepatic IRI by suppressing inflammatory reactions.This is consistent with previous studies which found that fisetin could alleviate I/R damage in other organs albeit through alternative mechanisms.Garg et al.[18] reported that fisetin reduced apoptosis,oxidative stress,and inflammation in myocardial infarction model by modulating PPAR-γas well as the NF-κB pathway.Zhang and Cui [25] also demonstrated that fisetin alleviated cerebral IRI by suppressing the NF-κB activity via inflammatory and antioxidant pathways.Besides,fisetin was reported to have protective effects in liver diseases.For instance,fisetin mitigated nonalcoholic fatty liver disease by suppressing the TNF-α/RIPK3 pathway mediated inflammation [26] .Our study now implicates the NLRP3 inflammasome as a critical target of fisetin in liver,and together with these prior studies highlights fisetin as a candidate for treating hepatic inflammation induced by different diseases.

Fig.5.Inhibition of AMPK by compound C (10 μmol/L) partially alleviates the anti-inflammatory ability of fisetin in vitro .A-C:RAW264.7 macrophages were treated as indicated (FIS-10;10 μmol/L),and production of IL-1 β,IL-18 and TNF-α was measured by ELISA;D-J:relative expression of p-GSK3 β (Ser9),p-AMPK (Thr172) and NLRP3 inflammasome-associated proteins in the cells from A .* P ＜ 0.05,** P ＜ 0.01,NS P ＞ 0.05.

Much literature involving hepatic IRI emphasizes the significance of the NLRP3 inflammasome,especially its critical role in maturation and release of detrimental cytokines [9,10,27].As expected,we observed the elevated expression of NLRP3 inflammasome-associated proteins in bothinvivoandinvitromodels,which coincided with the enhanced production of IL-1βand IL-18.Moreover,we found that fisetin suppressed the activation of the NLRP3 inflammasome and concomitant release of downstream inflammatory cytokines in hepatic IRI.Consistently,albeit in the context of a diabetic nephropathy model,it was found that fisetin supplementation reduced NLRP3 expression in high fat diet-fed mice [28] .

We also observed that GSK3βactivity was increased by I/R operation,manifested by decreased inhibitory phosphorylation expression of GSK3βat Ser9.Similar to previous reports,fisetin treatment suppressed GSK3βactivity through increasing p-GSK3β(Ser9) expression [17,29].GSK3βis documented to control glucose metabolism and inflammatory reaction [30,31],and may exert immune-regulatory effects by direct control of proinflammatory molecules,or by altering the cellular metabolism of immune cells to influence their activation or differentiation [11,32].In macrophages,toxins increase GSK3βactivity and activate NLRP3 inflammasome,while GSK3βinhibitors can suppress these effects [33] .Besides,inhibiting GSK3βmay protect against hepatic IRI by regulating antioxidant response and mitochondrial permeability transition [34] .Moreover,GSK3βinhibition contributes to elevated CREB activity and IL-10 production,as well as diminished NF-κB activity,which together can minimize inflammation and liver damage [11,35].

As a central regulator of cellular energy metabolism,activation of AMPK exerts anti-inflammatory effects in various inflammatoryrelated diseases [36-38].Notably,Qing et al.[39] showed that activating AMPK could suppress the NLRP3 inflammasome by boosting M2 macrophage polarization via mTOR.Wang et al.[40] also showed that suppressing AMPK/mTOR-mediated autophagy activates the NLRP3 inflammasome in Kupffer cells.Of relevance,fisetin was reported to activate AMPK/mTOR signaling in pancreatic cancer cells [41] .In this study,we confirmed that fisetin suppressed NLRP3 inflammasome through AMPK signaling in liver tissue and murine macrophages.Interestingly,the activation of AMPK in macrophages was documented to be regulated by GSK3β[11] .On the contrary,AMPK was also reported to regulate the activity of GSK3βin macrophages [42] .However,we observed that pharmacological inhibition of AMPK did not influence the inhibitory effects of fisetin on GSK3β,nor did it entirely reverse the effects of fisetin on the NLRP3 inflammasome.While our data firmly invoke a role for the GSK3β/AMPK pathway in the fisetin-mediated regulation of the NLRP3 inflammasome,the latter finding suggests that GSK3βinhibition is at least partially uncoupled from AMPK.This further suggests that other pathways must be involved.

Finally,we must acknowledge the limitations of our study.The pathophysiological process of IRI is complex and cannot be explained by inflammation alone.Moreover,the mouse model used a standardized I/R time and therefore more experiments are required to determine the extent of benefit provided by fisetin under different ischemia time that produce different degrees of IRI.Furthermore,there are other properties of fisetin that we have not considered in our analysis including antioxidant activity.

In summary,we validated that fisetin protects the liver against IRI.Additionally,fisetin suppressed GSK3β,accompanied by AMPK activation,leading to NLRP3 inflammasome suppression and diminished pro-inflammatory cytokines productions.This body of work suggests fisetin intervention as a new strategy to treat hepatic IRI.

Acknowledgments

None.

CRediTauthorshipcontributionstatement

Jun-LiangPu:Data curation,Formal analysis,Writing -original draft.Zuo-TianHuang:Data curation,Formal analysis,Writing -original draft.Yun-HaiLuo:Data curation,Software,Writing-review &editing.TongMou:Data curation,Software,Writing -review &editing.Ting-TingLi:Data curation,Methodology,Writing -review &editing.Zhong-TangLi:Data curation,Methodology,Writing -review &editing.Xu-FuWei:Writing -review &editing.Zhong-JunWu:Conceptualization,Funding acquisition,Supervision,Writing -review &editing.

Funding

This study was supported by grants from the National Natural Science Foundation of China (81672959,81873592 and 81703063),the Science and Technology Commission Foundation of Chongqing,China (cstc2019jscx-gksbX0005),the Natural Science Foundation of Chongqing,China (cstc2018jscx-msybX0133) and the graduate tutor team construction project of Chongqing Municipal Education Commission Foundation,China (dstd201801).

Ethicalapproval

This study was approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (2020-514).

Competinginterest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.