The protective effect of the natural compound hesperetin against fulminant hepatitis in vivo and in vitro - PubMed (original) (raw)
doi: 10.1111/bph.13645. Epub 2016 Nov 28.
Peixuan Yang 3, Qiaoling Zhou 1, Bozhi Cai 4, Manon Buist-Homan 2 5, He Cheng 1, Jiyang Jiang 1, Daifei Shen 1, Lijun Li 1, Xiajiong Luo 1, Klaas Nico Faber 2 5, Han Moshage 2 5, Ganggang Shi 1
Affiliations
- PMID: 27714757
- PMCID: PMC5341490
- DOI: 10.1111/bph.13645
The protective effect of the natural compound hesperetin against fulminant hepatitis in vivo and in vitro
Xueting Bai et al. Br J Pharmacol. 2017 Jan.
Abstract
Background and purpose: Liver diseases are mostly accompanied by inflammation and hepatocyte death. Therapeutic approaches targeting both hepatocyte injury and inflammation are not available. Natural compounds are considered as potential treatment for inflammatory liver diseases. Hesperetin, a flavonoid component of citrus fruits, has been reported to have anti-inflammatory properties. The aim of this study was to evaluate the cytoprotective and anti-inflammatory properties of hesperetin both in vitro and in models of fulminant hepatitis.
Experimental approach: Apoptotic cell death and inflammation were induced in primary cultures of rat hepatocytes by bile acids and cytokine mixture respectively. Apoptosis was quantified by caspase-3 activity and necrosis by LDH release. The concanavalin A (ConA) and D-galactosamine/LPS (D-GalN/LPS) were used as models of fulminant hepatitis. Liver injury was assessed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver histology and TUNEL assay and inflammation by inducible NOS (iNOS) expression.
Key results: Hesperetin blocked bile acid-induced apoptosis and cytokine-induced inflammation in rat hepatocytes. Moreover, hesperetin improved liver histology and protected against hepatocyte injury in ConA- and D-GalN/LPS-induced fulminant hepatitis, as assessed by TUNEL assay and serum AST and ALT levels. Hesperetin also reduced expression of the inflammatory marker iNOS and the expression and serum levels of TNFα and IFN-γ, the main mediators of cell toxicity in fulminant hepatitis.
Conclusion and implications: Hesperetin has anti-inflammatory and cytoprotective actions in models of acute liver toxicity. Hesperetin therefore has therapeutic potential for the treatment of inflammatory liver diseases accompanied by extensive hepatocyte injury, such as fulminant hepatitis.
© 2016 The British Pharmacological Society.
Figures
Figure 1
Structures of hesperetin and hesperidin.
Figure 2
Hesperetin has cytoprotective and anti‐inflammatory effects on primary rat hepatocytes. (A) Hesperetin reduces GCDCA‐induced caspase‐3 activation (GCDCA: 50 μmol·L−1). (B) Hesperetin prevents cleavage of PARP as assessed by Western blot. (C) GCDCA and CM do not induce necrotic death of hepatocytes. The inhibitory effect of hesperetin on GCDCA‐induced apoptosis and CM‐induced inflammation is not accompanied by an increase of necrosis as assessed by LDH leakage in supernatant of cultured hepatocytes. LDH release is expressed as % of total LDH content of hepatocytes. (D) The protective effect of hesperetin is not abolished upon inhibition of the p38 and ERK MAPKs and the PI3K pathway. (E) Hesperetin attenuates the inflammatory response of hepatocytes as assessed by iNOS mRNA determination by qPCR; All experiments were performed in duplicate wells and each experiment was repeated using hepatocytes from five different isolations; Values are mean ± SD, ns indicates not significant; *P < 0.05, significantly different from GCDCA or CM.
Figure 3
Hesperetin dose‐dependently protects against Con A‐mediated fulminant hepatitis. (A) Macroscopic appearance of livers indicating a beneficial effect of hesperetin to the dark surface of liver with passive congestion. (B) Haematoxylin–eosin staining of liver sections: in Con A hepatitis it shows hepatocellular degeneration (cloudy swelling), eosinophilic focus of cellular alteration with pale pink cytoplasm, condensed hypereosinophilic cytoplasm and shrunken nuclear occur spontaneously with one or two affected hepatocytes (arrows) and infiltration of inflammatory cells (mononuclear: lymphocytes and macrophages) (arrowheads), which are significantly reduced by hesperetin. Magnification 200X (upper panel), 400X (lower panel). (C) Quantitation of infiltrating leukocytes represented as average number of individual liver sections in Con A and Con A + Hesperetin groups. Magnification of measurement area is 400X. (D) Serum samples show increased total bilirubin levels in Con A‐treated animals, which is (partially) reversed by hesperetin. Samples represent (from left to right): Control, Con A, Con A and increasing doses of hesperetin. (E) Serum markers of liver injury, AST, ALT and total bilirubin, are significantly induced in Con A hepatitis. Hesperetin attenuates the rise in ALT, AST and TBil levels. (F) Serum levels of cytokines as assessed by
elisa
demonstrate a significant rise of IFN‐γ and TNFα in Con A hepatitis, which is reduced by hesperetin. Values are mean ± SD; Control group and Control Hesperetin group n = 8, Con A and Con A + Hesperetin groups n = 15, *P < 0.05, significantly different from Con A.
Figure 4
Apoptotic cell death and inflammation in Con A induced fulminant hepatitis are attenuated by hesperetin. (A) Apoptosis was assessed by TUNEL assay and visualized by Alexa Fluor 488: Hesperetin significantly reduced the number of TUNEL‐positive nuclei in Con A hepatitis; (B) Hesperetin attenuated Con A‐induced inflammation as assessed by iNOS Western blot analysis. GAPDH was used as a loading control. (C) Expression of cytokines in liver tissue was determined by qPCR and expressed as fold increase compared to control. Con A induced the expression of inflammatory and T‐cell derived cytokines. The induction of all cytokines, except Il‐1β, was attenuated by hesperetin. Values are mean ± SD; Control group n = 8, Con A and Con A + Hesperetin groups n = 15. *P < 0.05, significantly different from Con A.
Figure 5
Hesperetin reduces caspase‐3 activity in fulminant hepatitis. Hesperetin attenuates caspase‐3 activity in fulminant hepatitis. Hesperetin was administered to animals at 200 mg·kg−1. D‐GalN/LPS and to a lesser extent Con A, induced an increase in caspase‐3 activity, which was significantly attenuated by hesperetin in the D‐GalN/LPS model. Values are mean ± SD *P < 0.05, significantly different as indicated.
Figure 6
Hesperetin protect mice from D‐GalN/LPS induced fulminant hepatitis. (A) Macroscopic appearance of livers. (B) Haematoxylin–eosin staining of liver sections: In D‐GalN/LPS‐induced fulminant hepatitis, there is pale eosinophilic staining, absence of nuclear detail, nuclear fragmentation (arrows), patchy areas of blood and abundant apoptotic hepatocytes (arrowheads). Magnification: 200X (upper panel) and 400X (lower panel). (C) Quantitation of infiltrating leukocytes, represented as average number of individual liver sections in D‐GalN/LPS and D‐GalN/LPS + Hesperetin groups. Magnification of measurement area is 400X. (D) Serum samples show hyperbilirubinemia in D‐GalN/LPS ‐treated animals, which is not reversed by hesperetin. Samples represent (from left to right): Control, Hesperetin, D‐GalN/LPS, D‐GalN/LPS and increasing doses of hesperetin. (E) Serum markers of liver injury, AST, ALT and TBil are significantly induced in D‐GalN/LPS hepatitis. Hesperetin (Hst) attenuates the rise in ALT and AST levels but not in TBil levels. (F) Serum levels of inflammatory cytokines as assessed by
elisa
. D‐GalN/LPS induces a strong increase in TNF serum levels, which is attenuated by Hst. Values are mean ± SD; Control group and Control Hst group n = 8, D‐GalN/LPS and D‐GalN/LPS + Hst groups n = 15. ns: not significant; *P < 0.05, significantly different from D‐GalN/LPS.
Figure 7
Apoptotic cell death and inflammation in D‐GalN/LPS‐induced fulminant hepatitis are attenuated by hesperetin. (A) Apoptosis was assessed by TUNEL assay and visualized by Alexa Fluor 488. Hesperetin significantly reduced the number of TUNEL‐positive nuclei in D‐GalN/LPS induced hepatitis; (B) Hesperetin attenuated D‐GalN/LPS‐induced activation of JNK as assessed by Western blot analysis for phospho‐JNK. Total JNK and GAPDH were used as reference proteins. Right panel shows the quantitation of pJNK/JNK. (C) Expression of cytokines in liver tissue was determined by qRT PCR and expressed as fold increase compared with control. The inflammatory cytokines TNFα, IL‐1β and IL‐6 are induced in D‐GalN/LPS hepatitis, and this induction is attenuated by hesperetin. Furthermore, expression of FasL and TLR‐4, mainly expressed on inflammatory and immune cells, is increased in D‐GalN/LPS hepatitis, and this increase is attenuated by hesperetin. Values are mean ± SD; Control group and Control Hesperetin group n = 8, D‐GalN/LPS and D‐GalN/LPS + Hesperetin groups n = 15, *P < 0.05, significantly different from D‐GalN/LPS.
Figure 8
Potential mechanism of hesperetin in fulminant hepatitis. In acute and chronic liver injury, activation and infiltration of inflammatory cells (Kupffer cells, neutrophils) and death of functional hepatocytes occurs. Hesperetin protects hepatocytes from apoptotic cell death induced by TNFα/FasL, activating ‘death receptors’ and IFN‐γ, produced by T‐cells like NKT cells. Hesperetin has both anti‐inflammatory effects on inflammatory cells and protective effects on hepatocytes and these effects are independent. The anti‐apoptotic effects of hesperetin are in part due to reduced activation of the pro‐apoptotic MAPK JNK.
References
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