Exploration Of The Role Of Retinoic Acid Receptor Aluminum Induced Amyloid Deposits Pathway

Al is exiting widely in the natural environment and living environment, becauseof its physical and chemical properties, are widely used in daily life and production,thus increasing the chance of being accessed to our body. Many studies show that Alis a potent neurotoxicant and may contribute to the formation of amyloid deposits inthe brain, and Al can inhibit the key enzyme α-ADAM10enzyme secretion innon-amyloidogenic pathway. Retinoic acid receptor as effects of factors thatphysiological and biochemical from vitamin A in the body, its can control the neuralcell differentiation and growth and nerve cell repair, therefore its be consideredneurotrophic factor. This topic through two section include in vivo and in vitro toresearch Al are inhibiting α-ADAM10secreted enzyme by retinoic acid receptors,regulating non-amyloid deposits pathways.1. In vivo test section:Objective: Neurological damage model was established by sub-chronicintraperitoneal injection of Al(mal)3in rats, exploration of Al accumulation in thenervous system, and examine the mechanism whether Al regulate α-ADAM10receptors secretion of enzymes by RARs.Methods: Take40healthy male SD rats were randomly divided into four groups:experimental control (Al3+0μM/kg), the low-dose group (Al3+15μM/kg), mid-dosegroup (Al3+30μM/kg) and high-dose group (Al3+45μM/kg).Over two months,rats inthe saline group received daily intraperitoneal(i.p.)injection0.9%saline. Afterexposure, the rats were sacrificed by cervical dislocation, taking cortex and hippocampus separately stored at-80℃. Al content in brain was assayed by graphitefurnace atomic absorption spectrometry, to assay cortex and hippocampus ofRARα,RARβ,RARγ receptor,α-ADAM10secretion of enzymes and C83expressingby Western blot.Results: With the increasing of the dose of Al(mal)3exposure,Al(mal)3contents inbrian in Al(mal)3exposed groups were increased statistically along with theincreased of Al(mal)3exposure dosage, which was significantly higher than thosein saline group(P<0.05),Since the Al(mal)3can inhibit RARs receptorexpression,experiments show that Al(mal)3can make RARα receptor expression wassignificantly decreased,in cortical of RARα receptors was significantly compared tothe other group (P<0.05); but its only45μM/kg group was compared with othergroup were significantly (P<0.05) in rat hippocampus. RARβ receptors wasincreasing with exposure doses showed a downward trend in rat cortex andhippocampus, but only30μM/kg and45μM/kg group statistically significantcomparet with saline group(P<0.05), there was significant difference that45μM/kgdose group compare to other groups(P<0.05). RARγ receptors decrease with theincrease of the exposure dose, but only45μM/kg compared with the saline controlgroup have significant difference (P<0.05). In the rat brain cortex and hippocampus,since the RARα receptor can adjuste to the expression of the α-ADAM10enzymesecretion,experiments show that inhibiting the expression of RARα receptorsaffected by the Al.α-ADAM10protein expression decreased comparing with thecontrol group was significantly (P<0.05), in a dose-response relationship. Theprotein expression of C83in all Al(mal)3exposed group were increased statisticallythan other group (P<0.05).Conclusion: The rat subchronic exposure to Al(mal)3can lead to accumulation of Alin the brain, became the basis of Al cause nervous system toxicity;experiments show that inhibiting the expression of RARα receptors affected by the Al, which makesα-ADAM10secretion of the enzyme expression decreased;the expression of RARαis inhibited by Al,so that the secretion of α-ADAM10enzyme is inhibited,and thenα-ADAM10effects non-amyloid pathway in vivo.2. In vitro test sectionObjective: Establish two cell model to reserch the retinoic acid receptor pathway byal(mal)3exposure and adding agonists in PC12cells, exploring the mechanism whatAl whether to adjust the α-ADAM10enzymes secreted by RARs receptor.Method:1.PC12cells were divided into four groups,exposure with Al(mal)3to fourdoses0μM/l,100μM/l,200μM/l and400μM/l,using CCK8detect cell viability afterexposure of Al(mal)3;the cells were observed with an optical microscope afterexposure, the effact of Al(mal)3on cell morphology; using Western blot to assayRARα,RARβ,RARγ receptor,α-ADAM10secretion of enzymes and C83expressionin cells.2. The cells were divided into five groups, basis on the last part of the cellexperiments, select200μM/l as a control group exposed to Al(mal)3,adding retinoicacid receptor agonists RA regulation this pathways. Grouping cells:0uM/L,DMSO,RA,200μM/Land RA+200μM/L,after adding retinoic acid receptoragonists RA and200μM/l exposed to detecte cell viability by CCK8;the cells wereobserved with an optical microscope after exposure in five groups;using Westernblot to assay RARα,RARβ,RARγ receptor,secretion of α-ADAM10enzymeexpression and C83.Results:1.The cells model of Al exposureBy CCK8showed that: with the increase of Al (mal)3exposure dose,the inhibition rate of PC12cells were increased, cell viability was significantly reduced,and compared with0μM/Lgroup,100μM/L,200μM/Land400μM/L dose groups werestatistically significant (P<0.05). After testing the results of Western blot assayshowed: RARα, RARβ receptor,α-ADAM10and C83were decreased expression byincreases Al(mal)3exposure dose, the difference was statistically significant(P<0.05),a dose-response relationship. The RARγ receptor expression withincreasing exposure dose decreased, only400μM/ldose group were significantdifferences by statistical analysis (P<0.05).2. The model of adding agonist RACCK8results show that:Compared with0μM/l, DMSO group has nostatistically significant difference, Showed that the dose of DMSO on PC12cellmorphology no inhibitory activity; while in RA group,200μM/L group andRA+200μM/L group compared with the control group was significant difference(P<0.05), Showed that RA can increase cell viability,200μM/L Al(mal)3group willinhibit cell viability, RA+200μM/L compared with each groups were statisticallysignificant compared to others (P<0.05). Western blot assay showed that: AfterAl(mal)3exposure,RARs receptors was significantly(P<0.05)higher compared withthe other groups, The results showed that the expression of agonist RA can makeRARs receptors was significantly higher in normal PC12cells;In200μM/L exposuregroup RARα and RARβ receptor expression was significantly reduced，indicatingthat Al(mal)3inhibits RARα and RARβ receptor expression, and it’s compared withother groups was significant difference (P<0.05);In RA+200μM/L group, RARα andRARβ receptors results showed that the agonist can reduce Al(mal)3injury to them,and the difference was statistically significant (P<0.05).The result of α-ADAM10secretion of enzymes and C83:RA group was compared with the control and DMSOgroup, the related protein expression was significantly higher, and there was significant difference (P<0.05); in200μM/L exposure group:the expression ofα-ADAM10secretion of enzymes and C83significantly reduced, with a significantdifference (P<0.05); in RA+200μM/Lgroup show that Al(mal)3is reduceddamagment after adding agonists about α-ADAM10secreted enzyme and C83, andthe difference was statistically significant (P<0.05).Conclusion:1.PC12cells were exposed with different doses of Al(mal)3,the significance of theexperimental results is consistent with in vivo tests, it is necessary further illustratedby in vitro experiments,Al may be regulated the secretion of α-ADAM10enzyme bythe retinoic acid receptors RARα and RARβ in the dissolved Aβ pathway, then injuryof the nervous system.2.The cells model of Al exposure show that, RARs receptors and other relatedproteins occur significant meaningful damage when the concentration of200μM/L.So choose200μM/L dose Al(mal)3exposure and adding agonists RA forcomparison. The results showed that RA will adjust RARα and RARβ receptors toincrease the α-ADAM10enzyme secretion hydrolysis of APP, reducing theformation ofAβ and neurofibrillary tangles.