Research On Neuroprotective And Anti-inflammatory Effects Of Oleracein E And Extracts From Portulaca Oleracea L.

Portulaca oleracea L.is an annual herb in the family of Portulacaceae.The aerial part of P.oleracea is used as an edible vegetable and also a traditional Chinese medicine.It has many kinds of functions such as heat-clearing,detoxifying,dispelling heat from blood to stop bleeding.Modern pharmacological studies have shown that P.oleracea has a wide range of activities,including antibacterial,anti-inflammatory,hemostatic,anti-oxidatant,anti-aging,anti-anoxia,neuroprotective,hypoglycemic and hypolipidemic effects.For example,extracts from P.oleracea exhibit protective effects on neuronal damage induced by hypoxia,rotenone,D-galactose/NaNO2 and AlCl3;they also exert anti-inflammatory effects on several animal models such as ear swelling,foot swelling,cotton swab granuloma,ulcerative colitis,and eczema,as well as on LPS-activated macrophage cellular model.However,to date,the neuroprotective and anti-inflammatory principles in P.oleracea are largely unknown.Section 1:Research on neuroprotective activity of oleracein EOleracein E(OE)(8,9-dihydroxy-1,5,6,10b-tetrahydro-2H-pyrrolo[2,1-a]isoquinoline-3-one)is an alkaloid bearing a rare catecholic tetrahydroisoquinoline/pyrrolidone tricyclic skeleton,which was firstly isolated from medicinal plant P.oleracea.Our previous study found that OE(15 mg/Kg)exhibited neuroprotective effects on large dosage of D-galactose/NaNO2-induced dementia mice and rotenone-induced Parkinson's disease mice models,through scavenging free radicals,inhibiting lipid peroxidation,alleviating oxidative stress,inhibiting neuronal apoptosis,and inhibiting ERK phosphorylation in the midbrain and striatum.Whether OE can exert neuroprotective effects on AlCl3-induced dementia mice or NaNO2-induced memory consolidation disorder mice model is unknown.1.The mouse model with brain oxidative stress and neurotoxicity was established by intraperitoneal injection of AICl3(40 mg/Kg/day)for 28 days.Neuroprotective effect of OE(3 and 15 mg/Kg/day,i.g.,28 days)on AICl3-toxicated mouse model was firstly evaluated,from aspects of body weight,survival rate,cognitive function,organ index,Al contents in the brain and plasma,oxidative stress-related biomarkers,hippocampal histology and expression of apoptosis-related proteins.Nootropic drug piracetam(PA)was used as the positive control.Results showed that AlCl3 can significantly increase plasma and brain Al levels(p<0.001 and p<0.01),compensatorily upregulate brain GSH level(p<0.01),increase plasma and brain MDA contents(p<0.001,p<0.01)and result in oxidative stress.Some hippocampal neuronal cells were damaged,and expression of pro-apoptotic protein Caspase-3 in hippocampal CAl,CA3 and CA4 regions(p<0.001,p<0.001,p<0.001)and Bax in hippocampal CA1 and CA4 regions(p<0.01,p<0.001)were significantly increased.Unexpectedly,these alterations did not cause significant impairment in spatial learning and memory ability of AlCl3-toxicated mice(p>0.05).Ultraviolet spectrophotometric assay indicated that OE could form stable chelate complexes with Al ion.An increase in brain Al content by OE(15 mg/Kg)likely occurred through chelating with Al,which reduced the toxicity of free Al ion in the brain.OE at high dose significantly reduced the MDA content(p<0.05)by regulating the antioxidant biomarkers such as SOD,CAT and GSH,and alleviated the brain oxidative stress.Similar with PA,high and low dose of OE significantly inhibited the Caspase-3 expression in hippocampal CA4 and CA1 regions induced by AlCl3(p<0.05,p<0.001),however,they had no significant influence on Bax expression(p>0.05).Above results indicated that the phenolic alkaloid OE as an antioxidant,Al chelator and apoptosis inhibitor,can alleviate oxidative stress and neurotoxicity induced by AlCl3.2.Effect of OE(3 and 15 mg/Kg/d,i.g.,56 days)on Kunming mice exposed to NaNO2(90 mg/Kg/d,i.p.,56 days)was firstly evaluated.NaNO2 toxication can cause death in mice.On the 56th day,the survival rate in NaNO2 model was only 70%,the survival rate in PA group was 75%,while the survival rate in high dose OE group and control group was 85%,and survival rate in low dose OE group was 90%,there are no significantly differences among five group(p<0.05).Intraperitoneal injection of NaNO2 caused a significant increase in brain and plasma MDA(p<0.001,p<0.05),however,it didn't cause significant alteration in methemoglobin level(p>0.05).Although a few hippocampal neuronal cells were damaged after NaNO2 exposure,the learning and memory function of mice were not significantly impaired(p>0.05).Above results showed that intraperitoneal injection of NaNO2(90 mg/Kg/d,56 days)alone indeed cause oxidative stress in the brain,however,it was not enough to cause dementia mouse model.Compared with NaNO2 group,the positive drug PA significantly reduced the compensatory increase in brain SOD(p<0.05),but it failed to significantly reduce brain MDA level(p>0.05);low dose of OE significantly increased brain GSH content(p<0.05),although high dose of OE had no significant effects on SOD,CAT and GSH(p>0.05),both high and low dose of OE can significantly reduce brain and blood MDA levels(p<0.01,p<0.01;p<0.01,p<0.001).Concerning previous experiment indicated that OE possesses strong ability to scavenge DPPH free radical and ROS in vitro,it can be speculated that OE may decrease lipid peroxidation in the brain through directly scavenging ROS.Section 2.Research on anti-inflammatory effects of OE and extracts from P.oleracea on LPS-induced sepsis mice modelSepsis is a systemic inflammatory response syndrome caused by infection of pathogenic microorganisms into the body.Traditional Chinese medicines which possess heat-clearing and detoxifying function play the important roles in the treatment of sepsis,through antagonizing endotoxin and reducing inflammatory mediators.LPS,the cell wall ingredient of Gram-negative bacteria,is also known as endotoxin.LPS-induced inflammation in mouse RAW264.7 macrophage cells and sepsis mouse model have become important models for the study of anti-inflammatory drugs.Many studies demonstrated that extracts from P.oleracea can significantly inhibit the excessive release of inflammatory cytokines in LPS-activated RAW264.7 macrophage cell,such as TNF-a,IL-6 and NO.In addition,our previous study discovered that OE and other tetrahydroisoquinoline alkaloids have the ability to significantly inhibit the excessive release of NO in LPS-activated RAW264.7macrophage cell.However,to date,whether extracts and compounds from P.oleracea can exert anti-inflammatory activities on LPS-induced sepsis mouse model has not been reported.Therefore,in this paper,the anti-inflammatory effects of an ethnolic extract(PAAs)from P.olearcea(250,500,1000 and 4000 mg/Kg),a total organic acid fraction(Acid)from PAAs(50,100,400 and 1000 mg/Kg)and OE(5,15 and 45 mg/Kg)were evaluated for the first time on the sepsis mouse model which was established by single intraperitoneal injection of LPS(5 mg/Kg)for 8 h.The results indicated that,in comparison with the control group,LPS caused hypersecretion in the eyelids,spirit droop and movement reduction,simultaneously,LPS significantly increased plasma NO(p<0.001),TNF-a(p<0.001)and IL-6(p<0.001)levels,as well as plasma AST and ALT activities(p<0.001,p<0.01).(1)PAAs significantly decreased plasma NO in sepsis mice at 500-4000 mg/Kg(p<0.05)and significantly decreased TNF-a(p<0.05)and IL-6(p<0.05)levels at 1000?4000 mg/Kg,similar with the anti-inflammatory effect of the positive drug dexamethasone at 5 mg/Kg.(2)Compared with LPS-induced sepsis mice,Acid dose-dependently reduced plasma NO level(p<0.05)and IL-6 level(p<0.01)at 50?1000 mg/Kg,however,it had no significant influence on iNOS(p>0.05),in addition,it significantly decreased plasma TNF-a content(p<0.05)at 400?1000 mg/Kg,and significantly inhibited AST and ALT activities at 100?400 mg/Kg(p<0.05,p<0.01;p<0.01,p<0.001),thus alleviating the acute inflammatory response in sepsis mice.GC-MS analysis of methylated product of Acid preliminarily indicated that there were 8 main compounds in Acid fraction,and they were succinic acid,benzoic acid,malic acid,citric acid,4-hydroxycinnamic acid,ferulic acid,eicosanoic acid and heneicosanoic acid.(3)OE at 15?45 mg/Kg significantly inhibited the excessive release of NO in the plasma of sepsis mice(p<0.01),however,it had no significant influence on plasma TNF-a and IL-6 levels in sepsis mice(p>0.05).Concerning in vitro experiments indicated that PAAs,Acid,and OE all had a strong ability to directly scavenge NO2-,it is suggested that this scavenging ability may be closely related to their inhibiting abilities against NO overproduction in sepsis mice.Above results indicated that total organic acid is a bioactive fraction in P.oleracea which can relieve acute inflammatory reactions in LPS-induced sepsis mice.However,whether tetrahydroisoquinoline alkaloids with OE as the representative are anti-inflammatory compounds in this antipyretic and detoxifying medicinal plant P.oleracea,still need to be evaluated and corroborated.