| Objective: This study takes the MAPKs signal transduction pathway as the main clue,observes and verifies the a-beta 25-35 induced PC12 cells by preparing the drug-containing serum of rats and cultured the adrenal myelin osomal cells(PC12 cells)in rats.Injury has a protective effect,with MAPKs signal transduction pathway and inflammatory response as the starting point,from the molecular biological point of view to reveal the preventive protection mechanism of renal brain soup on nerve cell damage,for renal brain soup can alleviate Alzheimer’s disease can provide an effective scientific basis for the development of clinical drugs.Materials and methods:The first part,the preparation of rat-containing serum:Healthy SD rats were randomly divided into 8 groups: blank group,model group,high dose group,medium dose group,low dose group,Chinese medicine high dose,p38 inhibitor group,Chinese medicine high dose,ERK inhibitor group,Chinese medicine high dose and JNK inhibitor group,5 rats in each group.8 groups of rats at the same time in the same environment,timed morning and evening feeding,drinking water unlimited,in the second morning of feeding began to inject stomach medicine.Based on the adult clinical daily dose multiplied by the rat drug coefficient of 0.018,the dose concentration of chinese medicine high dose group(including inhibitor group)was calculated at 14.4g(Kg/day): the dose group of the medium dose group was given concentration of 7.2g(Kg/day): the low dose group was given 3.6g(Kg/day)for 5 consecutive days.Blank groups and model groups were not specially treated and were regularly fed for 5 days.All dose groups of Chinese medicine were the same except for the different concentrations of medicines contained.After the last administration of 1h,the abdominal anaesthetic,blood in the abdominal aorta,3000 centrifugation 10 min separation to obtain serum,56 degrees ℃ inactivated 30 min,sterile filtration,that is,drug-containing serum.Place in a4℃ refrigerator and keep for use.Part II,Cell Experiment:Rapidly melt the frozen PC12 cells in a water bath at 37 degrees℃,centrifugated,discarded waste liquid,placed in a DMEM medium with 10% FBS heavy suspension,inoculated in a culture bottle,placed in a 5% CO2,37℃ incubator culture 24 h,2 to 3d liquid exchange once.After the cells are fully fused,the waste liquid is discarded,observed and counted under an inverted microscope,inoculated in a new culture bottle at a density of 105cells/ml,and finally cultured in a CO2 thermostatic incubator.According to the experimental requirements,PC12 cells are divided into the following five groups(1)Blank group:(cultured 24 h with pre-prepared blank group rat serum)。(2)Model group:(adding a total of 24 h to a total of 24 h a beta 25-35 with a final concentration of 20umol/L)in both the rat serum and the final concentration of 20umol/L)(3)Low-dose group of Chinese medicine:(A beta 25-35 with final concentration of20umol/L and 24 h of drug-containing serum containing low dose of renal-brain soup)(4)Chinese medicine dose group:(a total concentration of 20 umol/L a beta 25-35 and a drug-containing serum containing a medium dose of renal brain soup cultured 24h)(5)Chinese medicine high dose group:(a total concentration of 20 umol/L a beta25-35 and a high dose of renal brain soup containing the drug-containing serum culture24h)The above Chinese medicine high,medium and low dose group(a final concentration of 20umol/L a beta 25-35 and containing a high,medium and low tonic brain soup of the drug-containing serum cultured 24h).All cells are cultured in incubators of 37 degrees ℃and 5% CO2.The content of leukocyte interleukin-8(IL-8),leukocyte interleukin 1 beta(IL-1 beta),leukocyte interleukin-6(IL-6)and nucleo-factor-NF-B(NF-B)were measured using ELISA.The m RNA levels of AKT and Nrf2 in cells were detected with PCR.Results:1.Rehydration of renal brain soup contains drug serum can protect A beta 25-35 induced damage PC12 cells2.Effect of renal brain soup on PC12 cell AKt/Nrf2 m RNA expression AKtm RNA expression is highest in the high-dose group,AKtm RNA expression is the lowest in the model group,and the expression of AKtm RNA is significantly reduced relative to the blank control group;Nrf2m RNA was the lowest expression in the model group,and Nrf2 m RNA in the high-dose group and inhibitor group had the highest expression.Compared with the blank control group,the expression of Nrf2 m RNA in the model group was significantly reduced.The expression of Nrf2 m RNA increased significantly in the cells of the high,medium,low,and high dose-inhibitor group.Compared with the low-dose group,Nrf2 m RNA expression increased significantly in the medium,high-dose,high-dose and inhibitor group.Compared with medium dose,Nrf2 m RNA expression increased significantly in the high-dose,high-dose-and-inhibitor group.The expression of Nrf2 m RNA in high-dose and inhibitor group cells was significantly higher than in the high-dose group.3.Effect of the drug-containing serum on the pc12-35 induced PC12 myocellular interleukin-6(IL-6)content of a beta 25-35 induced brain soup Of the five models,the high-dose-inhibitor group had the lowest expression of leukin 6 and the model group had the highest expression of leukin 6.Compared with the blank control group,the expression of interleukin 6 in the five model groups increased significantly.Compared with the model group expression of interleukin 6,high,medium,low,high and high in the inhibitor group,the expression of interleukin 6 in cells was significantly reduced compared with the low and medium dose group of high-dose-inhibitor groups.4.Effect of the drug-containing serum on the content of PC12 cell leukocyte interleukin-8(IL-8)induced by A beta 25-35.Of the five models,the high-dose and inhibitor group had the lowest expression of leukin 8 and the model group had the highest expression of leukin8.Compared with blank control group,the expression of low,medium,high-dose group interleukin 8 was significantly increased,and the 8 expression in low,medium,high dose,high dose and inhibitor group cell decreased significantly compared with the model group,and compared with the low,medium and high dose group and the high dose-inhibitor group,the expression of interleukin 8 in cell decreased significantly;5.Effect of the drug-containing serum on a beta 25-35 induced PC12 cell leukocyte interleukin 1 beta(IL-1 beta)content In the five models,the high-dose and inhibitor group had the lowest level of beta expression of leukun 1 and the model group had the highest amount of leukin 1 beta expression.Compared with the blank control group,the expression of leukade 1 was significantly increased compared with the blank control group,but compared with the model group,the expression of leukin 1 beta in each group decreased significantly,and the expression of leukocyl 1 beta in each group decreased significantly compared with the low,medium and high dose groups,and compared with the low,medium and high dose groups,the expression of leukocyl 1 beta in cell decreased significantly.6.Effect of the drug-containing serum on the content of PC12 cell nucleo-factor-Nf-B(NF-B)induced by A beta 25-35 In the five models,the high-dose and inhibitor group had the lowest expression of NF-KB-1,and the model group had the highest expression of the white interleukin NF-KB-1.Compared with the blank control group,the expression of NF-KB-1 in the low,medium,high dose,and high-dose inhibitor groups increased significantly.NF-KB-1 was less expressive than in the model group in low,medium,and high-dose,high-dose,high-inhibitor group cells.Conclusion:1.The renal brain soup contains a drug serum that protects PC12 cells that induce damage to A beta 25-35.2.Renrenal brain soup activates AKt/Nrf2 signaling pathway,AKt/Nrf2 m RNA expression increases,can inhibit the expression of pro-inflammatory cytokines,thus playing an anti-inflammatory anti-nerve cell apoptosis effect.3.The drug-containing serum of renal brain soup protects PC12 cells induced damage to A beta 25-35,and its mechanism may be by reducing the content of PC12 cell leukocyte interleukin-8(IL-8),leukocyte interleukin 1 beta(IL-1 beta),leukocyte interleukin-6(IL-6)content,reducing the toxic effect of A beta and reducing the damage to the nerves caused by inflammatory factors.4.The supplementary renal brain soup contains a drug serum that inhibits the expression of nuclear factor-NF-B(NF-B),suggesting that the protective effect of renal brain soup on A beta 25-35 induced PC12 cells may have the participation of NF-B signaling pathways. |
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