Curcuma Longa Extract On SK-N-SH Cell Damage Protection, Apoptosis And Related The Influence Of Protein Expression

Objective:Vascular dementia (VD) is a acquired intellectual damage syndromewhich by a series of cerebrovascular factors (ischemia or hemorrhagicand acute or chronic oxygen deficiency cerebrovascular disease) lead tobrain tissue damage of brain dysfunction. About the treatment of VD,there is no special method. Modern medicine is mainly cerebrovasculardisease prevention, for clinical have cerebrovascular disease risk factorsof patients should be early prevention and control to prevent theoccurrence of cerebral vascular disease and VD. Traditional Chinesemedicine for dementia record of the early, its unique theory system andrich medical Practice, for the treatment of the disease has providedvaluable experience. Literature has been reported Curcumin on ischemia-reperfusion rats have protection, the subject is Curcuma Longa Extract toSK-N-SH cell damage protection and Bcl-2, Bax genes and the influenceof the protein expression, this paper discusses the treatment of vasculardementia mechanism.Methods:1SK-N-SH cells culture method: SK-N-SH cells cryopreserved inliquid nitrogen, while use for recovery. In37℃,5%saturated humidityin-cubator of CO2training, pour out the culture medium when the cells ofthe cultivate bottle, Use D-Hank ’s liquid gently swings a wash cells,digestive2-3min. Abandon digestive juices, add in1640medium to endthe the digestion. Gently blowing make cells scattered open, viewspecimens under a microscope, dilute the cells with1640medium for5x105a/ml,vaccination in24hole training board continue to cultivate, we can be used for experiments when cells in full.2Will Curcuma Longa Extract dissolved in a little DMSO made upwith1x105μmol/L the mother liquor,-20℃freeze-stored, withcomplete medium diluted to the concentration when used, the content ofDMSO is less than0.1%.3The experiment is divided into five groups, respectively for thecontrol group, the model group,20μ mol/L Curcuma Longa Extractgroup,40μ mol/L Curcuma Longa Extract group and80μ mol/LCurcuma Longa Extract group.4Curcuma Longa Extract on SK-N-SH cells glutamic acid (Glu)damage the influence of protection.Curcuma Longa Extract on SK-N-SH cells Glu damage modelprotection: take cells covered with single the24hole training board, suckabandon the original medium, use D-Hank’s liquid wash cells once, eachhole to join1640liquid1ml, and then respectively to join the CurcumaLonga Extract of20μ mol/L,40/μ mol/L,80μ mol/L. The training of anhour after join Glu, continue to cultivate8h,16h,24h. The controlgroup does not add Glu damage.5Outcome5.1Morphology observation is under a microscope SK-N-SH cellgrowth and the change shape each group.5.2By MTT method to determine the cells with vigorIn the above damage occurs after the end of different time (8h,16h,24h), join MTT (0.5mg/ml end concentration), continue to cultivate3hours, suck to medium, each hole to join dimethyl sulfoxide (DMSO)200ul, stay hole particles is completely dissolved, move into the96hole inthe plate, by using enzymes standard instrument (λ=492nm) determinethe light density values (OD).5.3Lactate dehydrogenase (LDH) activity determinationAccording to the above damage occurs continue to cultivate16hafter collecting the cultivation clear fluid, according to reagent box manual determination of LDH activity.6Curcuma Longa Extract on in Glu after injury SK-N-SHextracellular SOD, MDA content influenceAccording to the above damage occurs continue to cultivate16hafter collecting the cultivation clear fluid, according to reagent boxmanual determination of SOD, MDA content.7Curcuma Longa Extract on Glu damage SK-N-SH cell cultureafter the influence of cell apoptosis7.1Western blot assay test Bcl-2, Bax Protein expression.7.2RT-PCR method to detect Bcl-2mRNA, Bax mRNA expressionlevel.Results:1Morphological observation: SK-N-SH cells have obvious injuryunder the500umol/L Glu action, Under a microscope，cells protuberantdis-appear, swelling round shrinkage, refractive index of decline, stickwall function drops, cell aggregates phenomenon is obvious, some cellswere broken into pieces; Give Curcuma Longa Extract all can ificantlyreduce Glu to SK-N-SH cell morphology change effects and performancefor the cell refraction is better, cell debris formation decreased cellaggregation reduction.2MTT method for determination of the cell vitality and Gluneurotoxicity cause SK-N-SH cell damage inhibition rate: After treatmentby Glu, the reduction MTT reduced, OD value was significantly lowerthan the control group (P<0.01), SK-N-SH cells Glu neurotoxicitydamage model of OD value, three Curcuma Longa Extract dose groupwas higher than the model group,8h three dose groups in the inhibitionrates were6.2%,30.7%,60.9%,16h three dose groups in the inhibitionrates were20.3%,42.4%,78.4%,24h three dose rates were suppressed,19.8%,41.8%,67.3%, curbing the Glu damage, indicating that the drugthe best in the role of16h.3In the culture medium of LDH leaks out of the quantity and the inhibition rate: After16h by Glu injury, compared with control group, themodel group release a quantity of LDH significantly increased(P<0.01).Three Curcuma Longa Extract dosage groups (20μ mol/L,40μmol/L,80μ mol/L) to SK-N-SH cells Glu neurotoxicity damage model ofLDH leaks out quantity significantly reduced, and comparing the modelgroup compared have obvious difference (P<0.01), inhibit the release ofLDH, the inhibition rate of the three drug groups were43.5%,57.8%,76.0%, show that Curcuma Longa Extractcan significantl improve the cellvitality.4Curcuma Longa Extract on Glu induction by SK-N-SHextracellular SOD, MDA content changes: After16h, compared withcontrol group, model group SOD energy declined obviously, MDAcontent increased significantly (P<0.01), show that caused by theneurotoxicity Glu SK-N-SH cell injury free radicals can appear themetabolic disorder. Curcuma Longa Extract each dose group of SODactivity of increased significantly, and reduce the MDA level, and modelgroup has more obvious difference (P<0.05or P<0.01), curbing the Gludamage.5Glu neurotoxicity cause SK-N-SH cells after injury of Bcl-2, Baxexpression influence: Through the Western blot test detection Bcl-2, BaxProtein expression: Compared with control group, Bcl-2protein expres-sion significantly reduced and the Bax protein expression significantlyincreased in model group, indicating that the model was successful;compared with model group, each drug group with Curcuma LongaExtract the increase of concentration, Bcl-2protein expression levelincreases, and Bax protein expression level is reduced. Through the RT-PCR method for the detection of Bcl-2and Bax content in the SK-N-SHcells: Compared with control group, Bcl-2mRNA content signifi-cantlydecreased, and Bax mRNA levels increased significantly in model group(P<0.01); Compared with the model group,Curcuma Longa Extract roleof Bax and Bcl-2transcription Product have obvious change, Bcl-2 mRNA content increased and Bax mRNA content decreased (P<0.05orP<0.01), it explains the Curcuma Longa Extract on Bcl-2expression isup-regulated and on Bax expression is down-regulation, inhibiting cellapoptosis.Conclusion:1Glutamic acid can cause SK-N-SH cell damage;2Curcuma Longa Extract on SK-N-SH cell glutamate injury hasaprotective effect;3Curcuma Longa Extract treatment for vascular dementia hascertain curative effect, it is by improving the cell activity, scavenging freeradicals, inhibiting cell apoptosis realize.