| Purpose: In our country, with advances in technology and the continuousdevelopment of the population's aging process, non-infectious diseases aredoing increasingly serious harm to people's health. Frequent cerebrovasculardisease increases the incidences of vascular dementia (of Vascular Dementia,VD) at a great rate, and the cases of vascular dementia increases sharply withage, therefore, VD is paid more and more attention in recent years. VD is themajor complications of cerebrovascular disease. It not only brings long-termpain to the patient himself or herself and impact on their quality of lifeseriously, but also creates a heavy burden to the entire family and the society.At present, there is no effective therapy on the VD treatment home andabroad. To prevent the cerebrovascular disease and VD, modern medicinedeals with patients with potential cerebrovascular disease risk mainly by earlystage primary prevention. For VD patients, besides active treatment ofcerebrovascular disease, they are often given calcium antagonist drugs,cholinesterase inhibitors and other drugs to control their activity andpsychiatric symptoms in order to slow the progression of the disease, alleviateits symptoms and improve the patients' quality of life. Therefore, giving fullplay to the overall concept of Chinese medicine and the advantages ofdiagnosis and treatment will be very significant to the treatment of vasculardementia.This research is completed under the guidance of my tutor professor WangSiping and cell biological experiments had been done for this research,which is based on the basic theory of Traditional Chinese Medicine and theresearch takes the model of hypoxic injury of the nerve cells through Na2S2O4's damage to the human neuroblastoma cell tumor SK-N-SH. By studying theinfluence of turmeric extract crystals (including curcumin, curcumin go methoxy, double de-methoxy curcumin) on the SK-N-SH cell's damage andapoptosis, this research explored the effective parts of turmeric----turmericextract's hypoxic injury protective effect on SK-N-SH cells and its mechanismand disucussed the mechanism of the treatment of vascular dementia in orderto provide theoretical basis for clinical medication.Methods: Cell biology is used in this experiment, and it is divided intofive groups: control group, model group,20μmol L-1turmeric extract group,40μmol L-1turmeric extract group,80μmol L-a turmeric extract group.20μmol L-1turmeric extract group,40μmol L-1turmeric extractgroup and80μmol L-1turmeric extract group get drugs pre-protection1h,then sodium dithionite (Na2S2O4) is added, both are given hypoxic injury.Make the final concentration at5mmol/L and continue to culture themfor16h,24h,32h, then observe the cell morphology, lactate dehydrogenase(LDH) activity measurement, then use the MTT method to test the cells'survival rate, and test the superoxide dismutase (SOD) activity andmalondialdehyde (MDA) production in the cell culture medium; and detectcell caspase-3activity by Western blotting method.Results:1. Morphological observation Observe SK-N-SH cell growth andmorphological changes of each group under inverted phase contrastmicroscope.The results showed that: Under the inverted phase contrast microscope,the shape of SK-N-SH cells were round, oval, spindle, or irregular with fewprotruding, among them most are round. The SK-N-SH cells are adherent cells,with strong refraction ability, and the cells grow into a cluster at a fairly fastrate after3h's culture. Under the effect of5mmol/L Na2S2O4, the SK-N-SHcells are obviously damaged. The cells decreased in number, the protrusionsdisappeared, the swellings had a round shrinkage, the refractive indexdecreased, the adhering ability reduced, the cell body gradually decreased, thebody particles gradually increased, and we can see most of the cells had died,some cells were broken into pieces. After given turmeric extract, the morphological changes of SK-N-SH cells under hypoxic environment couldbe significantly reduced, with better refraction, less cell fragments, and thecell body gradually increased and cell body particles were gradually reduced.2. MTT method cell viability determinationThe results showed that: Control group8h MTT value of0.77±0.03,the model group was0.12±0.01, low dose group,0.22±0.01, the middledose group was0.27±0.01, the high dose group was0.34±0.03; controlgroup16hMTT value of0.83±0.040.19±0.01, the model group value of0.19±0.01, low-dose group was0.25±0.01, the middle dose group was0.31±0.01, high-dose group was0.41±0.02; the MTT value of control groupfor24hours1.13±0.04,0.19±0.01in the model group, low dose group,0.25±0.010.34±0.01, the middle dose group, the high dose group was0.44±0.03,the OD value of the group with three doses of turmeric extract (20μmol L-1,40μmol L-1,80μmol L-1) was significantly higher than that of themodel group, the difference was significant(P <0.01).3. Determination of lactate dehydrogenase (LDH) activityThe results showed that: the LDH values of each group are as follows: ofthe control group, it is370.61±35.96U/L; of the model group, it is1000.77±16.78U/L; of the low dose group, it is735.52±44.0U/L; of the mediumdose group, it is571.31±37.00U/L; of the high dose group, it is467.34±91.23U/L. There are significant differences (P <0.01) compared with themodel group, turmeric extract inhibited the LDH release, and the increase ofthe dose reduces the LDH release. They are negatively correlated.4. The impact of turmeric extract on SOD, MDA that produced in thesupernatant afte hypoxic SK-N-SH.The results showed that: the SOD values of each group are as follows: thecontrol group14.62±0.41U/L, the model group10.62±0.43U/L, the lowdose group12.86±0.30U/L2, the medium dose group13.71±0.27U/L, thehigh dose group13.99±0.38U/L. SOD value: the control group3.39±0.33U/L, the model group6.11±1.04U/L, the low dose group4.08±0.63U/L,the medium dose group3.79±0.42U/L, the high dose group3.63±1.15U/L. Compared with the normal control group, the level of MDA was significantlyincreased (P <0.01), while the level of SOD significantly decreased(P <0.01)in the cell culture medium. Compared with the model group, turmericextract of low, medium and high density group can significantly decrease theMDA level (P <0.01) in SK-N-SH cells in the cell culture medium, while thelevel of SOD was significantly increased (P <0.01), and turmeric extract caninhibit SK-N-SH cells hypoxia damage.5. The effects of turmeric extract on hypoxia-inducible SK-N-SH's damageof model cell Caspase-3activity:By Western blot method to detect the expression of Caspase-3protein:compared with normal control group,Compared with the normal control group,sodium dithionite(Na2S2O4)induction cell Caspase-3increased significantly;compared with the sodium dithionite(Na2S2O4)induction group, turmericextract of low, medium and high density can significantly decrease theSK-N-SH cells Caspase-3activity.Conclusion:1. Turmeric extract protects SK-N-SH cells from hypoxic injury2. The protective effect of turmeric extracts of SK-N-SH cells from hypoxicinjury through antioxidant effects and inhibition of apoptosis..3. The study provides theoretical basis for the turmeric extract treatment ofvascular dementia. |
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