| ObjectiveMangroves are woody plant communities dominated by mangrove plants that occur in high salinity soil and periodic impregnation,and have special structures and functions.The main component of mangroves is Sonneratia which has high medicinal and edible value.Thereinto,Sonneratia apetala,belonging to the Sonneratia family,is commonly used to treat diseases such as hepatitis,sprains,bleeding,and diarrhea.Its fruits are edible,contain pectin,and are used as medicine and food.Modern pharmacological studies have shown that Sonneratia apetala fruits have anti-oxidative,anti-inflammatory and liver protective activities.And the previous research found that its hepatoprotective effect might be related to the polysaccharides it contains.In recent years,many polysaccharides from natural products have been widely used for the prevention and treatment of liver injury due to their good protective effects and small side effects.As the main place of substance metabolism and detoxification in the body,liver is vulnerable to drugs,toxic substances and alcohol,causing liver cell necrosis or apoptosis,liver tissue structure changes,and eventually liver injury.With the gradually increasing clinical medication species and doses,the frequency of drug-induced liver injury has increased.Therefore,looking for drugs that can prevent and treat liver injury has become the focus of many medical researchers.The liver injury caused by overdose of acetaminophen(APAP)accounted for the largest proportion of clinical cases in drug-induced liver injury.Modern pharmacological studies were likely to seek effective therapeutic drugs for liver injury through APAP-induced liver injury model.Hence,the experiment intended to study the preliminary characterization of Sonneratia apetala fruits polysaccharides and evaluated its antioxidative activity through antioxidant activity experiments in vitro,and then explored its hepatoprotective effect and underlying mechanism on acetaminophen(APAP)induced liver injury mice.Methods1.Preliminary characterizationThe water extraction and alcohol precipitation method was used to extract polysaccharides from Sonneratia apetala fruits,and the DEAE-52 cellulose and Sephadex G-100 chromatography columns were used to separate and purify the Sonneratia apetala fruits polysaccharides.And the carbonhydrate,uronic acid,sulfate and protein contents of polysaccharides isolated from Sonneratia apetala fruits were determined.Then FT-IR analysis was also used to analyze the preliminary structures of polysaccharides.2.Evaluation of antioxidant activity in vitroThe scavenging abilities of Sonneratia apetala fruits polysaccharides on DPPH,superoxide anion and hydroxyl radicals as well as the reducing ability were measured to evaluate its antioxidatant activities in vitro.3.Protective effect and mechanism of SAP on APAP-induced liver injuryICR mice were randomly divided into 6 groups:normal control group(NC),model group(APAP),positive drug group(NAC),and SAP groups(100.200,and 400 mg/kg).Each group was administered by gavage,and the mice weights were recorded before each administration.Except for NC and APAP group being given corresponding volumes of deionized water,the other four groups were given the corresponding drugs at a dose volume of 0.1 mL/10 g once a day for 30 consecutive days.Eight hours after the last administration,except for NC group being intraperitoneally injected with saline,mice in each group were intraperitoneally injected with 220 mg/kg APAP saline solution to establish liver injury model caused by APAP.After fasting for 12 h,the mice were weighed again.After taking blood,they were sacrificed and the liver was dissected out,weighed,recorded.and calculated the liver index.Liver tissue was cut from the same part of each mouse’s,fixed with paraformaldehyde.and then HE stained for pathological evaluation.Serum was separated and the serum transaminase ALT and AST levels were measured using biochemical kits.In order to further study the potential mechanisms of SAP on APAP-induced liver injury mice,the liver tissue was homogenated to determine the GSH,MDA content and GSH-Px.SOD activities using biochemical quantitative methods,to measure the TNF-α and IL-6 content using ELISA assays,to analyse the CYP2E1,GCLC,GiCLM.HO-1.NQO1,TNF-α and Ⅱ-6 mRNA levels using PCR technology,and to detect the CYP2E1,JNK.P-JNK,Caspase 3.Cleaved-Caspase 3,Bcl2.Bax,Nrf2.Keap1,GCLC,GCLM.HO-1 and NQO1 protein expressions using Western-blot method.Results1.Sonneratia apetala fruits crude polysaccharide(SAP)from Sonneratia apetala fruits,and then processed by DEAE-52 cellulose and Sephadex G-100 chromatography column to purify its three fractions SAP1,SAP2 and SAP3,of which SAP1 and SAP2 were the main components of SAP,accounting for more than 98%of its content.The results of chemical analysis revealed that the carbohydrate,uronic acid,sulfate and protein contents of SAP 1 and SAP2 were 68.75%and 56.15%,19.65%and 40.40%,4.52%and 2.30%,7.08%and 1.00%,respectively.Both of them were acidic polysaccharides with high levels of uronic acid.Thc FT-IR results showed that both SAP1 and SAP2 had the characteristic absorption peaks of polysaccharides and the glycosidic bond type was α-pyranosidc bond.SAP1 may mainly contain a-glucopyranosc,α-galactopyranosc,α-pyran mannose and α-xylanopyranosc,SAP2 may mainly contain α-glucopyranose and α-xylanopyranose.2.Sonneratia apetala fruits polysaccharides had good antioxidant activities in vitro,which could effectively remove DPPH,superoxide anion and hydroxyl radicals,and had certain reducing ability.The antioxidant activity in vitro of SAP was stronger than its two fractions.3.After APAP being injected,compared with NC group,the liver index and serum transaminase levels of each group mice increased significantly,indicating that the liver injury model was successfully established.After treatment with drugs,compared with APAP group,SAP could significantly reduce liver index,serum ALT and AST levels,alleviate liver pathological damage,increase GSH content,GSH-Px and SOD activity,reduce MDA content and inflammatory factor TNF-α and IL-6 levels;enhance GCLC,GCLM,HO-1 and NQO1 mRNA expression and inhibit CYP2E1 mRNA expression;In addition,SAP could inhibit JNK phosphorylation as well as expressions of CYP2E1,apoptosis-related proteins(Cleaved-Caspase 3,Bax)and Nrf2 signaling pathway-related proteins(cytoplasmic Nrf2 and Keap1),while promoted Caspase 3,Bcl2,GCLC,GCLM,HO-1,NQO1 and nuclear Nrf2 expressions.ConclusionIn summary,Sonneratia apetala fruits polysaccharides mainly composed of two acidic polysaccharides SAP1 and SAP2 whose glycosidic bond is α-pyranoside bond,wherein SAP1 is mainly composed of α-glucopyranose,α-galactopyranose,α-pyran mannose and α-xylopyranose,SAP2 is mainly composed of α-glucopyranose and α-xylopyranose.In vitro antioxidant activity experiments had verified that SAP have good antioxidant activities;APAP-induced liver injury experiments in vivo showed that SAP had protective effect on APAP-induced liver injury,and its mechanism might be related to the improvement of the body’s antioxidant capacity,the inhibition of oxidative stress,inflammatory response,apoptosis,and the regulation of CYP2E1 expression as well as the Nrf2 signaling pathway. |
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