The Protective Effect Of Dihydromyricetin-encapsulated Liposomes On Exhaustive Exercise-induced Liver Inflammation By Orchestrating Macrophage Polarization And Its Underlying Mechanisms

BackgroundIt is well-known that regular and suitable exercise is beneficial to keeping and promoting health.However,severe exercise or exhaustive exercise(EE)can cause multiple organ tissue damage,including muscle and liver,mainly manifested by inflammation through oxidative stress.There are no effective prevention and treatment measures.Recent studies have found that regional immune regulation plays an important role in the process of liver inflammation.Liver macrophages are important participants in immune regulation.Under the influence of the microenvironment,liver macrophages can polarize to form two polarization phenotypes:proinflammatory(M1,classical activated)and anti-inflammatory(M2,selective activated).Macrophages with two phenotypes jointly regulate the process and outcome of liver disease.Therefore,precise regulation of liver macrophages polarization is expected to become a key step in preventing and treating exercise induced liver inflammation.Dihydromyricetin(DHM)is a flavonoid compound that mainly exists in the stems and leaves of Ampelopsis grossedentata(known as rattan).The content of DHM in the dry stems and leaves of rattan is as high as 30%.It has a variety of protective effects such as anti-oxidative stress,anti-tumor,and improving glucose and lipid metabolism.It can also protect from inflammatory damage of the liver induced by carbon tetrachloride,ethanol,and other chemicals and thus play a protective role in the liver.Other studies have found that DHM can regulate macrophage and polarization thus preventing atherosclerosis via inhibiting cholesterol accumulation and inflammasome activation.However,it is not clear whether it has a protective effect on EE-induced liver injury.Besides,DHM has problems of rapid metabolism,which greatly limits its hepatoprotective effect.Therefore,it is urgent to find effective means to prolong the working duration of DHM.In recent years,with the development of materials science and interdisciplinary integration,a variety of controlled release drug delivery systems have shown great prospects in improving bioavailability.Liposomes,as a simple and efficient drug delivery system,are in the spotlight.Long-circulating liposomes can also be constructed by grafting chemical or biologically inert polymers onto the liposome,which can resist the influence of the microenvironment and prolong the duration.At present,many studies have reported that liposome delivery systems can improve the bioavailability of plant compounds and effectively prolong the action time of plant compounds.However,the dihydromyricetin-encapsulated liposomes delivery system and its application in the protection of exercise-induced liver injury have not been reported.Objective1.To confirm the protective effect of DHM on exercise-induced liver injury;2.To construct liver accumulated DHM liposome(DHM-Lipo)and reveal the slow-release effect,distribution in vivo and the roles on EE-induced liver injury;3.To elucidate the role of DHM-Lipo in regulating phenotypic polarization of macrophages in the inflammatory microenvironment;4.To reveal the mechanism of SIRT3/HIF-1α signaling pathway in DHM regulation of hepatic macrophage phenotypic polarization.Methods1.A mice model of liver inflammation was established by exhaustive swimming exercise(referred to EE-induced liver injury).After 3 weeks of DHM intervention with 2,4and 8 mg/kg·BW daily,then we evaluated whether DHM has the effect of alleviating EE-induced liver injury by testing body weight,liver index,pathological index,liver function and the expression of inflammatory factors in serum and liver.To evaluate whether DHM can reduce EE-induced liver injury,and determine the lowest effective dose of DHM intervention.On this basis,the frequency of DHM administration was reduced to once every three days to observe whether DHM still has anti-inflammatory effects after prolonged intervention interval,and to explore the effect of prolonged DHM administration interval on its protective effect on EE-induced liver injury.2.We constructed DHM-Lipo and evaluated the physical and chemical properties of it,such as morphology,particle size,sustained release in vitro and biocompatibility.And we compared the inhibitory effects of DHM-Lipo and DHM on EE-induced hepatitis under the same intervention conditions(once every three days)through in vivo experiments.Furthermore,DHM fluorescence liposome(DHM-Lipo@IR-808)was constructed by grafting fluorescence groups for in vivo NIR fluorescence imaging,and the biodistribution of DHM-Lipo was detected by fluorescence reflectance imaging(FRI)ex vivo scans.3.The aggregation effect of DHM-Lipo on liver macrophages in mice with EE-induced liver injury was detected by the tracer of DHM-Lipo@IR-808 combined with immunofluorescence assay,and the regulation effect of DHM-Lipo on the polarization phenotype of liver macrophages was explored.LPS was used to stimulate primary liver macrophages and mouse RAW264.7 macrophages to establish a macrophage inflammation model.The regulation of DHM on phenotypic polarization of liver macrophages was further explored by flow cytometry in the inflammatory microenvironment.4.SIRT3 specific inhibitor and HIF-1α stabilizer were used to reveal that the role of SIRT3/HIF-1α signal pathway on that DHM regulated the polarization of macrophages under inflammatory environment in vivo and in vitro tested by the transcriptome,qRT-PCR and Western Blot.Results1.DHM can effectively inhibit EE-induced liver injure,but the hepatoprotective effect of DHM is significantly reduced by prolonging the DHM administration interval.The mouse model of EE-induced hepatitis was successfully constructed.The liver inflammatory infiltration was intensified,the blood biochemical indexes such as AST and ALT were abnormal,and the expression of inflammatory factors such as TNF-α,IL-1β and IL-6 in serum and liver tissue were significantly up-regulated.DHM(2、4、8 mg/kg·BW/d)treatment daily for 3 weeks can effectively inhibit inflammatory damage in mice.However,when the intervention interval of DHM(2 mg/kg·BW/d)was extended to once every three days,the anti-inflammatory and liver protection effect of DHM was significantly weakened,indicating that the problems of rapid metabolism and short residence time of DHM limited its anti-exercise hepatitis effect.2.DHM sustained-release delivery system based on liposomes can play a lasting role in inhibiting EE-induced liver injury.A nanoscale DHM sustained-release delivery system(DHM-Lipo)with stable physical and chemical properties and high biocompatibility was successfully constructed.Compared with the DHM group,DHM-Lipo can play a significant anti-inflammatory and liver protection effect stably under the condition of prolonged administration interval.The fluorescent liposomes DHM-Lipo@IR808 were synthesized by labeling DHM-Lipo with near-infrared fluorescent dye IR808 to realize DHM-Lipo in vivo tracing.DHM-Lipo was mainly distributed in liver in both in vivo and in vitro imaging.These results indicated that DHM-Lipo can release DHM sustainably and play a more lasting anti-inflammatory effect through accumulating in the liver.3.DHM-Lipo can aggregate in liver macrophages and further regulate the polarization of the immunophenotype from pro-inflammatory(M1)to anti-inflammatory(M2).A combination of DHM-Lipo@IR808-based tracing and immunofluorescence staining further revealed that DHM-Lipo could effectively accumulate in liver macrophages.In the macrophage inflammation model in vitro,flow cytometry and other tests showed that DHM could inhibit the expression level of M1-type markers and up-regulate the expression level of M2-type markers,indicating that DHM could accumulate in liver macrophages and regulate the polarization of macrophages from M1-type to M2-type.4.DHM regulates the phenotypic polarization of macrophages through SIRT3/HIF-1αsignaling pathways.DHM interferes with macrophage inflammation model.Transcri ptomic analysis showed that DHM has a significant anti-inflammatory effect and the key molecules of metabolic regulation SIRT3 and HIF-1α are important differential genes.In vitro and in vivo experiments,the expression of SIRT3 was significantly inhibited in the inflammatory model group,while the expression of HIF-1α was increased.DHM-Lipo and DHM intervention increased SIRT3 expression and decreased HIF-1α expression level.Further,after treatment with SIRT3 inhibitors and HIF-1α stabilizer,combined with transcriptomic analysis,qRT-PCR,WB,and immunofluorescence detection,it was found that DHM could inhibit glycolysis by activating SIRT3/HIF-1α signaling pathway,thus inhibiting macrophages from M1-type polarization.ConclusionIn this study,we found that DHM can inhibit EE-induced liver injury in mice,but rapid metabolism limits the lasting anti-inflammatory effects of DHM.DHM-Lipo,a slow-release delivery system of DHM constructed with liposomes,prolongates the anti-inflammatory time of DHM through the aggregation and slow-release effects of liver tissue and liver macrophages.DHM-Lipo inhibit macrophage glycolysis and regulates the transformation of macrophages from pro-inflammatory M1 type to anti-inflammatory M2 type,thus playing a role in regional immune regulation and inhibiting EE-induced hepatitis.In addition,SIRT3/HIF-1α signaling pathway is the key molecular mechanism of DHM inhibition of M1-type polarization in liver macrophages.This study provides a new idea for the prevention and treatment of EE-induced hepatitis by DHM,and also lays an experimental foundation for the study on the regulation of liver regional immunity by DHM.