| Objective: Aplysin,a member of bromine-sesquiterpene family,is a liposoluble compound extracted from red alga(Laurencia tristicha).In our laboratory,multiple biological activities from aplysin,including antioxidant,anti-inflammatory,anti-tumor and immunopotentiation were demonstrated.Furthermore,preliminary data indicated protective effects against ethanol-induced hepatoxicity,which was associated with modulation of mitochondria mediated apoptosis.Based on previous studies,the current study further investigated the hepatoprotective effects of aplysin and potential underlying mechanism,focusing on the TLR4 signaling pathway and intestinal flora.Methods:1.Evaluation of Aplysin induced hepatoprotective effects in rats with chronic alcohol-induced liver damage1)Animal grouping and model establishment: Sixty male Wistar rats(eight weeks old,180-220 g)were randomly assigned into three groups with random number table method:control group,ethanol-model group and aplysin treatment group.In the ethanol-model group,the rats were exposed to 56 degree wine 8 ml/kg/day via gavage for two weeks,then 12ml/kg/day via gavage for six more weeks.Rats in aplysin treatment group received identical ethanol exposure,along with 150 mg/kg/day aplysin pretreatment via gavage one hour prior to ethanol exposure for the whole eight week experiment.Rats in control group received identical volume saline gavage daily throughout the experiment.12 h post the last gavage,stool samples were collected with metabolic cage;animals were weighed and anaesthetized with 7% chloral hydrate,blood samples were collected from abdominal aorta,liver and intestinal tissues were collected for morphology assessment.(2)Histopathology on liver tissues: Liver tissues were fixed with 10% formaldehyde,processed for histology and sectioned.Hematolyxin and eosin(HE)staining was applied for visualization of the pathological changes.Transmission electronic microscopy(TEM)was utilized for the ultra structural alterations.(3)Hepatic function assessment: Five hepatic function enzymatic endpoints were assessed,including serum activity levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),choline esterase(CHE)and gamma-glutamyl transpeptidase(GGT).ALT and AST activities were quantified with Reitman method;ALP activity was calculated with PNP colorimetry;CHE activity was measured with hydroxylamine-ferric chloride colorimetry and GGT activity was determined with IFCC Rate Method.2.Effects of Aplysin on phagocytic capacity of Kupffer cells and TLR4 signaling pathway in rats with chronic alcohol-induced liver damage1)Primary Kupffer cells were isolated with in situ type IV collagenase perfusion through portal vein method.Isolated Kupffer cells were purified with Percoll density gradient centrifugation and cultured in petri dishes at 37 °C and 5 % CO2 environment.2)The identity of Kupffer cells was confirmed routinely by immunohistochemical staining for ED2,and Giemsa counterstaining was used to observe the morphology of Kupffer cells.Additionally,the phagocytic activity of Kupffer cells was determined with the ink phagocytosis test.MTT was utilized to test Kupffer cell activity.3)The plasma level of endotoxin was tested with Tachypleus Amebocyte lysate.4)In Kupffer cells,the m RNA expression levels of crucial molecules in TLR4 signaling pathway,including CD14,TLR4 and NF-κB were investigated with RT-PCR.5)The protein expression levels of CD14 and TLR4 in liver tissue was detected with Western Blot.6)The levels of TNF-α and IL-1β were detected with ELISA.3.Effects of Aplysin on intestinal floras in rats with chronic alcohol-induced liver damage1)Histopathological assessment of intestine: HE staining was used to observe the pathological changes in intestinal epithelium,and TEM was utilized to observe the ultra structural changes in epithelial cells.2)Intestinal flora genome DNA extraction: DNA affinity column and specific Inhibit EX reagent,along with specialized buffer system were used to extract intestinal flora genome DNA from faecal samples.3)Quantitative real-time PCR was used to quantify the changes in 16 s r DNA V3 variable zones of Escherichia coli,Enterococcus faecalis,bifidobacteria and lactobacillus in the samples.4)Potential correlations between the amounts of the four specific intestinal flora strains and hepatic function indexes were analyzed with SPSS software.Results1.Evaluation of Aplysin induced hepatoprotective effects in rats with chronic alcohol-induced liver damage1)HE staining revealed remarkable inflammatory cell infiltrations around central vein with apparent steatosis and Mallory bodies in ethanol-model group livers.In contrast,normal lobular architecture and hepatocytes radiating around central veins were present in samples from control or aplysin treatment rats without remarkable steatosis and inflammatory cells infiltrations.Further morphology assessment with TEM revealed more information:Dilatation of endoplasmic reticulum and large amount of lysosomes were observed in the model group,while smooth nuclear membrane and pores along with morphologically normal cytoplasm,rough endoplasmic reticulum and mitochondria were present in control and aplysin treatment groups.Small quantities of lipid droplets were present in aplysin treatment group.2)Hepatic function tests indicated significant elevation of serum ALT,AST,ALP,CHE and GGT activities in samples from model group comparing to those from control group(P<0.05).Meanwhile,aplysin treatment resulted in significant decrease of ALT,AST,ALP,CHE and GGT activities relative to control(P<0.05).2.Effects of Aplysin on phagocytic capacity of Kupffer cells and and TLR4 signaling pathway in rats with chronic alcohol-induced liver damagePrimary cultured Kupffer cells showed typical shuttle or irregular traiangle shapes.Giemsa staining demonstrated typical “fried egg” appearance.The purity was estimated to be more than 97%,as assessed by immunohistochemical staining with ED2.Ink phagocytosis test revealed that Kupffer cells from ethanol treatment group acquired significantly fewer carbon particles than those from the control group.Aplysin treatment effectively restored the phagocytic activity of Kupffer cells relative to ethanol treatment group.Compare with the normal control group,MTT results revealed that Kupffer cell activity in model group was significantly increased.In Aplysin-treated group,Kupffer cell activity was obviously decreased,compare with model group(P<0.05).After 8 weeks treatment,plasma endotoxin in the ethanol-treated group was significantly increased 20%relative to that in the control group(P<0.05).Administration of Aplysin significantly abolished the elevation of plasma endotoxin relative to the ethanol-treated group.The expression of CD14,TLR4,and NF-κB m RNA in Kupffer cells,as measured by RT-PCR,were significantly increased(by 1.1-,1.7-,and 2.0-fold,respectively,P<0.05)in the ethanol group relative to the control group(CD14/β-actin,0.32 ± 0.03;TLR4/β-actin,0.29 ± 0.03;NF-κB/β-actin,0.22 ± 0.02).The m RNA levels of CD14,TLR4,and NF-κB in the Aplysin treatment group were significantly lower(by 38 %,20 %,and 23 %,respectively)relative to those in the ethanol group.The protein expression levels of CD14 and TLR4 in alcohol-model group were increased 1.25-and 1.13-fold,compare with the control group.The protein expression levels of CD14 and TLR4 in Aplysin-treated group were decreased by 23% and 20%,compare with the alcohol-model group.The concentrations of TNF-α and IL-1β in the culture media of Kupffer cells,as measured by ELISA,were significantly increased(by 2.1-and 1.7-fold,respectively),in the ethanol group relative to those in the control group(TNF-α,21.86 ± 1.53 ng L-1;IL-1β,22.32 ± 1.19 ng L-1).The concentrations of those cytokines in the Aplysin administration group were significantly reduced(by 39 % and31 %,respectively)compared with those in the ethanol group(P <0.05).3.Effects of Aplysin on intestinal floras in rats with chronic alcohol-induced liver damageUnder optical microscope,no significant changes were observed between intestine tissues from control or aplysin groups,while apparent atrophic intestinal villus was observed in ethanol-model group tissues.Further assessment of morphology with TEM revealed widen cell junctions,sparse microvillus and increased amount of lysosomes in samples from ethanol-model group.In the Aplysin treatment group,microvillus structures become overall normal,but large quantities of lysosome were still present.The amount of Escherichia coli in model group was remarkably increased than that in control group(P<0.05);the amount of Enterococcus faecalis exhibited same trend but not statistically significant(P>0.05).Meanwhile,aplysin treatment reverted such changes.However,the amounts of Lactobacillus and Bifidobacterium were both sharply decreasing in the model group than that in the control group(P<0.05).With the pretreatment of Aplysin,the amounts of Bifidobacterium significantly increased relative to ethanol-model group(P<0.05).Similar trend was observed for Lactobacillus but not statistically significant(P>0.05).Correlation analysis between the four intestinal flora strains and five hepatic function enzymatic index revealed strong positive correlation between the amount of Escherichia coli and the activity levels of serum ALT.Conclusion1.Aplysin induced hepatoprotective effects in ethanol-exposed rats,as indicated by hepatic histopathological observations and hepatic function enzymatic assays.2.Aplysin enhanced the phagocytic capacity of Kupffer cells,increased the elimination of endotoxin;thus inhibited the activation of TLR4 signaling pathway in Kupffer cells,decreasing the expression levels of CD14,TLR4 and NF-κB and the concentration of downstream inflammatory cytokines TNF-α and IL-1β.3.Aplysin exerted protective effects in the intestine of ethanol-exposed rats.Quantitative analysis indicated that aplysin induced hepatoprotective effects are associated with its modulation to intestinal flora.Escherichia coli are likely to be the key bacteria involved in aplysin-induced intestinal flora modulation and subsequent protective effects against ethanol-induced hepatotoxicity. |
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