| Objective:At present,the main drugs for the treatment of Alzheimer’s disease(AD)are cholinesterase inhibitors(donepezil,galantamine)and NMDA receptor antagonists(memantine).These drugs mainly improve the symptoms of AD and cannot fundamentally stop or reverse the progression of AD.According to the clinical manifestations of AD,Traditional Chinese Medicine(TCM)has classified it into the category of "Dementia" in Traditional Chinese Medicine.TCM has accumulated rich and-valuable experience in the treatment of Dementia throughout the ages.In recent years,there have been a number of clinical studies on the treatment of AD based on syndrome differentiation of traditional Chinese medicine,and the results show that Traditional Chinese Medicine is effective in treating AD.Reviewing these clinical studies,it was found that most of the therapeutic methods studied were based on kidney tonifying in traditional Chinese medicine.This study included these literatures for Meta-analysis to systematically evaluate the efficacy and safety of kidney tonifying in AD treatment,providing evidence-based medical evidence for the treatment of AD by kidney tonifying in traditional Chinese medicine.At present,there is no specific drug for AD,and the candidate drug developed for Aβ,the main pathological factor of AD,has almost failed.Epidemiological studies have found that the incidence of AD in western developed countries is on the decline,which is due to the importance western developed countries attach to the protection of cerebral vascular health.In recent years,in the autopsy of AD patients,it was found that in addition to neuronal cell damage,there was also extensive capillary endothelial damage.Therefore,from the perspective of cerebral vessels,the protection of the blood-brain barrier,transport to clear the brain Aβ can be regarded as a new research idea.Professor Ru Guran drew up the prescription of "tonifying kidney and tonifying essence" to treat AD patients with definite curative effect in clinical practice.In the previous in vitro study,our research group not only confirmed that the serum of this prescription had protective effect on neurons,but also further confirmed that the decoction of the single drug rehmannia also had protective effect on nerve cells.However,5-hydroxyfurfural(5-HMF)is the effective component of rehmannia glutinosa.Therefore,this study investigated the effect of 5-hmf on the injury of Aβ1-42 oligomer induced in vitro blood-brain barrier(BBB)model of AD and its possible mechanism,so as to provide experimental data support for the treatment of AD by prepared rehmannia glutinosa.Method:Meta analysis:7 major databases were searched,including CNKI,Wan Fang(WF),VIP,CBM,PubMed,EMbase and web of science.Subject words and synonyms corresponding to PICOS were found according to the titles and principles of PICOS,and relevant literatures were collected in the past 10 years.Literature was screened by title,abstract and full text,and those that met the inclusion criteria were selected to extract relevant effective data.Review Manager5.3 software and Stata 12 software were used for Meta-analysis of the research results.Experimental part:1.CCK-8 method was used to screen the concentration of nearly 50%inhibition rate(IC50)of the bEnd.3 cell injury induced by Aβ1-42 oligomer.2.MTT method to screen the safe and effective concentration of 5-HMF:MTT method to detect the effect of 5-HMF at different concentrations on the activity of bEnd.3 cells induced by oligomer of Aβ1-a2·3.Tunel combined with DAPI staining was used to observe the apoptosis of bEnd.3 cells induced by 5-HMF on Aβ1-42 oligomer of a serum.4.Expression of apoptotic proteins(Bax,Bcl-2,Cyto-c,cleaved caspase3,caspase-3,p-PARP,PARP),tight junction proteins(ZO-1,Occludin,Claudin-5)and transporters(LRP-1,Pgp)were detected by Western Blot.Results:1.A total of 11 randomized controlled clinical studies were included in this Meta-analysis for systematic evaluation,and the evaluation results were as follows:the TCM kid-nourishing group was superior to the control group in clinical efficacy,TCM syndrome efficacy,improvement of MMSE score,ADAS-cog score and ADL score(P<0.05).In terms of safety,7 of the 11 included clinical studies had adverse reactions,and the symptoms were mild and relieved by themselves.The adverse reactions were included in the Meta-analysis and no statistical difference was found between the kidney tonifying group and the control group(P>,0.05),suggesting that the kidney tonifying method of Traditional Chinese Medicine may be safe and effective in the treatment of AD.2.CCK8 test results:the inhibition rate of oligomer of Aβ1-42 from 5uM,10uM,20uM,40uM and 80uM Aβ1-42 oligomer to the activity of bEnd.3 cells was close to 50%,compared with the Aβ1-42 oligomer group of model Aβ1-42,the difference was statistically significant(P<0.001).3.MTT assay results:(1)5-HMF within the concentration range of 1uM～400uM had non-toxic and damaging effects on bEnd.3 cells;(2)luM,lOuM and 50um5-HMF inhibited the damage of Aβ1-42 oligomer to bEnd.3 cell activity,and the differences were statistically significant(P<0.01).4.The results of Tunel combined with DAPI staining showed that compared with the control group,the number of cells in model Aβ1-42 oligomer group were increased by red fluorescence staining,and the nuclear volume was also shrunk and decreased.After the intervention of 5-HMF at different concentrations,the number of cells stained by red fluorescence decreased,and the number of nuclear volume shrinkage also decreased.5.Western Blot results:(1)expression of apoptosis-related proteins:After the treatment of bEend.3 cells with Aβ1-42 oligomer,the ratio of pro-apoptotic proteins Cyto-c/β-actin,Bax/Bcl-2,cleaved caspase-3/caspase-3 and p-PARP/PARP in the model Aβ1-42 oligomer group increased,with statistically significant differences(P<0.01).After the addition of 5-HMF intervention,their ratio decreased with the increase of 5-HMF concentration,with statistically significant differences(P<0.01).(2)expression of tight junction protein:after treatment of bEnd.3 cells with Aβ1-42 oligomer,the ratio of model Aβ1-42 oligomer ZO-1/β-actin,Occludin/β-actin,Claudin-5/β-actin decreased(P<0.05).After 5-HMF intervention,the ratio of 5-HMF low-dose group to medium-dose group increased with the increase of 5-HMF concentration,and the difference was statistically significant(P<0.05).However,ZO-1/β-actin and Claudin-5/β-actin in the high-dose group decreased with the increase of concentration(P<0.05),but were still higher than those in the model group.(3)expression of transporters:after treatment with Aβ1-42 oligomer,the ratio of LRP-1/β-actin and Pgp/(3-actin of model Aβ1-42 oligomer of bEnd.3 cells decreased,and the ratio increased with the concentration of 5-HMF after intervention.All the differences were statistically significant(P<0.01),but the increase of Pgp/β-actin was not significant.Conclusion:1.Through Meta-analysis,it was concluded that TCM kidney-tonifying method could improve the memory,cognitive function and daily living ability of AD patients without obvious adverse reactions.However,due to the limited number of included literatures,the total number of included patients was small and the literature quality was not high.Therefore,we should take an objective view of the results of this Meta-analysis.2.A certain concentration of 5-HMF can protect the normal physiological function of BBB from the damage of Aβ1-42 oligomer by protecting the activity of vascular endothelial cells,inhibiting apoptosis,and protecting the tight junction protein between endothelial cells and transporters on the cell membrane. |
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