Clinical And Basic Study Of The Clearing Heat And Detoxification Method In The Treatment Of Alzheimer’s Disease Based On The Pathogenesis Of "Toxin Damaging Brain Collaterals"

Objective:Through the theoretical discussion,the pathogenesis of AD in Western medicine and the etiology mechanism of traditional Chinese medicine were sorted out,and the pathogenesis of "Toxin Damaging Brain Collaterals" of AD was organically combined with the pathogenesis of AD neuroinflammation,which provides a theoretical basis for the prevention and treatment of AD by the method of clearing heat and detoxification（Huanglian Jiedu decoction）.Through clinical study,the clinical efficacy of the method of clearing heat and detoxification（Huanglian Jiedu decoction）on the overall cognitive function,mental behavior symptoms,overall impression,daily living ability and TCM syndrome changes in moderate AD patients were systematically evaluated,and it was confirmed that the method of clearing heat and detoxification（Huanglian Jiedu decoction）was an effective program for AD treatment.At the same time,it highlights the academic thinking of AD integrative medicine that combines disease and symptoms,staging and dialectics.Through basic study,the role of "Toxin Damaging Brain Collaterals" in the process of AD pathological change was explored,and the possible mechanism of action of the method of clearing heat and detoxification（Huanglian Jiedu decoction）on AD treatment was preliminarily elucidated,which provides an experimental basis for the prevention and treatment of AD traditional Chinese medicine,and also provides a new idea for the prevention and treatment of related central nervous system degenerative diseases.Methods:1.Clinical study methods:This trial was designed as a randomized controlled,open-label,noninferiority clinical study,and it referred to the Chinese Clinical Biology Model AD Operational Standard（BWG-2（2017））to formulate research exclusion standards.49 subjects were treated continuously for 24 weeks（the treatment group was treated with Huanglian Jiedu decoction,and the control group was treated with donepezil）and followed up for 36 weeks.The evaluation of the effectiveness of Huanglian Jiedu decoction in the treatment of moderate AD by primary/secondary clinical efficacy indicators（MMSE and CDR-SB/ADL and NPI）.To explore the clinical changes of TCM symptoms in subjects with AD through TCM syndrome indicators（CGIC-S）.The safety index evaluated the safety of Huanglian Jiedu decoction.2.Basic study methods:2.1 Experiment one:BV2 microglia were cultured in vitro,BV2 microglia growth curves were plotted by CCK-8 method,different concentrations of lipopolysaccharides（LPS）（0.25,0.5,1,2,4 μg/m L）were used to induce BV2 microglia for 24 h,BV2 microglia morphological changes were observed by inverted phase contrast microscopy,BV2 microglia activity and NO content were detected by CCK-8 method and Griess method respectively,TLR4 was detected by WB and q PCR,respectively.Expression of My D88 m RNA and protein.Subsequently,LPS-induced BV2 microglia with a concentration of 1μg/m L was selected for 24 h,and the concentration of TNF-α and IL-1βin BV2 microglia were detected by ELISA.2.2 Experiment two:LPS-induced BV2 microglia activation was used to establish an in vitro neuroinflammation model,and Griess method was applied to detect the inhibitory effect of nitric oxide（NO）produced by the drug-containing serum of Huanglian detoxification decoction on the in vitro neuroinflammation model.Real-time quantitative PCR（q PCR）was used to detect serum containing Huanglian Jiedu Decoction on the m RNA expression of TLR4,My D88,NF-κB p65,IKBa and COX-2 in LPS-induced BV2 microglia;Western blot was used to detect effects of serum containing Huanglian Jiedu Decoction on the protein expression of TLR4,My D88,NF-κB p65,p-NF-κB p65,IKBa,p-IKBa and COX-2 in over-activated BV2 microglia;ELISA was used to detect the effect of serum containing Huanglian Jiedu Decoction on the secretion of inflammatory cytokines TNF-α,IL-1β and IL-6 in an in vitro neuroinflammatory model.Results:1.Clinical study:During the course of the study,11 subjects were removed,and finally 25 subjects in the treatment group and 24 subjects in the control group were included in the statistical analysis.（1）12 and24 weeks after treatment,the MMSE and CDR-SB were significantly different in both groups compared with those before treatment（P<0.05）;and 36 th week,the MMSE and CDR-SB were not significantly different from those before treatment（P>0.05）;（2）At the 24 th week during treatment,ADL,NPI and caregiver distress scores were significantly different in both groups compared with those before treatment（P<0.05）;and ADL,NPI and caregiver distress scores were significantly different in the treatment group compared with those in the control group（P<0.05）;（3）At the 24 th week during treatment,the scores of yang hyperactivity,virulent fire,phlegm turbidity and blood stasis syndrome were significantly different in both groups compared with those before treatment（P<0.05）;and the scores of yang hyperactivity and virulent fire were significantly different in the treatment group compared with those in the control group（P<0.05）;（4）During the course of the study,no serious adverse reactions occurred in either group of subjects.2.Basic study:2.1 Experiment one:The results of CCK-8 and Griess method showed that BV2 microglia was in logarithmic growth phase at the 2-3d after subculture;LPS could significantly reduce the survival rate of BV2 microglia and increase the release of NO（P < 0.05）,showing a dose-response relationship within the experimental dose range.The results of Western blot and q PCR showed that the expression of TLR4 and My D88 m RNA and protein increased first and then decreased with the increase of LPS concentration,and reached the peak at LPS 1μg/m L（P < 0.05）.The results of ELISA showed that LPS（1μg/m L）significantly increased the concentration of TNF-α and IL-1β,and the difference was statistically significant（P<0.05）.2.2 Experiment two:Different concentrations of serum containing Huanglian Jiedu Decoction（5%,10%,20%）inhibited the production of inflammatory factors NO,and the inhibitory effect was in a concentrationdependent manner in the experimental concentration range.LPS induced BV2 microglia to overexpress the m RNA expression of TLR4,My D88,IKBa,NF-κB p65 and COX-2,while serum containing Huanglian Jiedu Decoction could effectively inhibit the m RNA expression of the above signal molecules.At the same time,LPS induced excessive activation of BV2 microglia to increase the protein expression of TLR4,My D88,p-NF-κB p65,p-IKBa and COX-2,and to reduce the protein expression of NF-κB p65 and IKBa,while the pretreated serum containing Huanglian Jiedu Decoction could inhibit the protein expression of TLR4,My D88,p-NF-κB p65,p-IKBa and COX-2,and increase the protein expression of NF-κB p65 and IKBa.In addition,the concentration of inflammatory cytokines TNF-α,IL-1β and IL-6 secreted by LPS-induced BV2 microglia significantly increased,while the secretion of the above inflammatory factors significantly reduced after serum containing Huanglian Jiedu Decoction was pretreated.Conclusion:1.The pathogenesis of AD neuroinflammation coincides with the AD ‘Toxin Damaging Brain Collaterals’ pathogenesis theory:neuritic plaques（NP）formed by Aβ deposition,neurofiber tangles formed by excessive phosphorylation of Tau protein in neurons,and pathological products with neuronal toxicity produced by microglia-mediated neuroinflammatory all belong to the category of‘endogenous toxicity’.2.Treatment with regular Huanglian Jiedu Decoction or donepezil significantly improved the overall cognitive function and overall impression of moderate AD patients with the yang hyperactivity or virulent fire as the main TCM syndrome,and there was a time effect on the therapeutic efficacy of both drugs,and no trend was seen to prevent or reverse the progression of the AD course during the after drug discontinuation follow-up period.3.The administration of regular Huanglian Jiedu Decoction or donepezil can significantly improve the daily living ability and mental behavior symptoms of moderate AD patients with the yang hyperactivity or virulent fire as the main TCM symptoms,and the therapeutic efficacy of Huanglian Jiedu Decoction was better than donepezil.4.The treatment of regular Huanglian Jiedu Decoction can significantly reduce the severity of the yang hyperactivity,virulent fire,phlegm turbidity and blood stasis syndrome in patients with moderate AD,and in reducing the therapeutic efficacy of the yang hyperactivity and virulent fire in patients with moderate AD,Huanglian Jiedu Decoction was significantly better than donepezil.At the same time,during the current study period,the subjects received no serious adverse reactions when they received Huanglian Jiedu Decoction or donepezil,and the participants took Huanglian Jiedu Decoction or donepezil with good compliance.5.Lipopolysaccharides（LPS）may induce BV2 microglia activation through the TLR4 signal transduction pathway to construct an in vitro neuroinflammation model,where 1μg/m L LPS was the optimal concentration for constructing a highly efficient and stable in vitro neuroinflammation model.6.The method of clearing heat and detoxification（Huanglian Jiedu decoction）may inhibit hyperactivation of microglia through the TLR4/NF-к B signal transduction pathway,improve the neuroinflammation response in the brain and exert neuroprotective effects.