| Objective: Alzheimer's disease(AD)is a progressive neurodegenerative disease with insidious onset and chronic property.Main clinical manifestations of AD are memory confusion,misrecognition,and changes in personality and behavior.The typical characteristics of AD are senile plaque(SP)formed by the deposition of ?-amyloid(A?)in brain tissue and neurofibrillary tangles(NFT)formed by hyper phosphorylation of Tau protein.Previous researches showed that Chinese medicine gardenia contain many active ingredients in its extract.Main components of gardenia are geniposide and crocins.These components possess various pharmacological activities such as promoting neuronal regeneration,anti-inflammatory,anti-apoptosis and anti-tumor.Comparative investigations of pharmacologic activities of these two components in AD,however,have remained to be addressed.Therefore,this investigation would aim to extract the geniposide and crocin-1 fractions from Chinese gardenia and employ 3×Tg-AD mice to demonstrate which components of gardenia are mainly responsible for anti-AD activity of this herbal remedy in vitro and in vivo.Methods: 1.Preparation and analysis of GE,TIG and TC in gardeniaGardenia was ground to a coarse powder and extracted with 60% ethanol.Then the fraction was concentrated,and freeze-dried to yield Gardenia Extract(Gardenia Extract,GE).GE was subjected to HPD-100 macroporous resin,and eluted with 25% ethanol and 60% ethanol,respectively,and then freeze-dried to yield total iridoid glycosides(TIG)and total crocins(TC).UPLC and LC-MS analysis was performed to identify major chemical components and determine content of geniposide and crocin-1 in three fractions.2.Protective effects of geniposide and crocin-1 for hippocampal primary neurons from 3×Tg-AD micePharmacological model of primary hippocampal neurons was employed to compare biological activities of geniposide and crocin-1 against AD.Hippocampus was removed from new born 3×Tg-AD mice.Neurons of hippocampus were prepared by plating,planting,digestion,and then cultured for 7 days.Geniposide and crocin-1 are administered on the 8th day.Cellular proteins were extracted for Western Blot measurement of A?1-42,Tau S396,GSK-3? and Bcl-2 proteins.3.Mitigating activitys of GE,TIG and TC in 3×Tg-AD mice Twenty-eight 4-month-old male 3×Tg-AD mice were randomly divided into 4 groups with 7 mice in each one: control,GE,TIG and TC.GE,TIG and TC are administered by gavage to mice daily at a dose of 100 mg/Kg for 8 weeks.At week 9,Morris water maze test was performed.Escape latency,space navigation and space exploration experiments in Morris water maze were performed to comparatively assess biological activities of TIG and TC.HE staining was performed to evaluate morphological changes of neurons in the hippocampus of mice.Expression of APP,Tau S396,A?1-42,BACE1,GSK-3?,Bcl-2 and BAX proteins in hippocampus were measured by Western Blot.Moreover,IHC was performed to evaluate expression of A?1-42,Tau S396,and APP proteins in hippocampus of brain tissue.ELISA was performed to assess levels of inflammatory cytokines TNF-? and IL-1? in serum to evaluate anti-inflammatory effects of components on Alzheimer's disease.In summary,behavioral experiments,pathology,molecular biology and immunology were employed to systematically evaluate the main components of anti-AD activities in gardenia.Results: 1.The results of UPLC and LC-MS analysis showed that main components of GE were geniposide and crocin-1,and contents were 33.4% and 15.1%,respectively.Main component of TIG was geniposide,and content was 62.2%.In addition,main component of TC was crocin-1,and content was 42.4%.2.Pharmacological experiment results of hippocampal primary neurons showed that expression levels of A?1-42 protein(P<0.01)and Tau S396 protein in hippocampal neurons of 3×Tg-AD mice were significantly decreased after administration of geniposide(P<0.05).Moreover,expression level of GSK-3? protein,which is responsible for A? deposition and Tau protein phosphorylation,was also significantly decreased(P<0.01),and expression level of bcl-2 protein was up-regulated(P<0.05).After administration of crocin-1,expression of protein A?1-42 and Bcl-2 was regulated if compared with model group(P<0.05).However,Tau S396 and GSK-3?,which are associated with Tau phosphorylation,remained no statistical difference(P>0.05).3.Morris water maze test results showed escape latency of both TIG and TC groups was significantly reduced if compared with the 3×Tg-AD model group(P<0.05).In addition,original platform residence time increased significantly(P<0.05)in treatment groups.Number of crossing original platform increased,but statistical difference was not observed(P>0.05).The results of HE staining showed that cells in the GE,TIG,and TC groups were tightly arranged,uniformly colored,and darkly stained cells were reduced if compared with the 3×Tg-AD model group.The immunohistochemistry results showed that expression of A?1-42 protein,Tau S396 protein and APP protein in GE group,TIG group and TC group were all decreased if compared with the 3×Tg-AD model group(P<0.5).Western-blot results showed that after administration of TIG and TC in 3×Tg-AD mice,the expression of APP protein was significantly decreased(P<0.01).If compared with 3×Tg-AD model group,A?1-42 and Tau S396 proteins in the hippocampus of mice and expression of GSK-3? which is associated with Tau phosphorylation in hippocampus of TIG and TC groups decreased significantly(P<0.05).Additionally,PP2 A were also upregulated(P<0.05).Bcl-2 expression levels significantly increased(P<0.05),and expression of pro-apoptotic factor BAX was significantly decreased(P<0.05).Proteins expression of BACE1 decreased in administration groups if compared with the 3×Tg-AD model group.However,P<0.05 was only observed in TC group.Furthermore,ELISA results showed that inflammatory cytokines TNF-? and IL-1? were significantly decreased in GE,TIG and TC groups(P<0.05).Conclusion:1.Geniposide and crocin-1 were successfully enriched into TIG and TC,respectively by HPD-100 macroporous resin.2.Geniposide and crocin-1 could reduce A? expression and inhibit neuronal apoptosis in primary hippocampal neurons of 3×Tg-AD mice.Geniposide and crocin-1 possess significant in vitro anti-AD pharmacological activities.3.TIG and TC extracts from gardenia could improve cognitive ability and memory function of 3×Tg-AD mice.Three fractions inhibited neuronal apoptosis and serum inflammatory response in AD mice.A? expression and tau protein phosphorylation in hippocampus of 3×Tg-AD mice could be inhibited.Therefore,GE,TIG and TC have anti-AD pharmacological activities.The content of geniposide and crocin-1 in three fractions of gardenia determine anti-AD activities of gadenia.4.TIG and TC could be used as the main quality control indicators in the future analysis of gardenia anti-AD drugs.Therefore,this maiden attempt demonstrated that CM gardenia is promising for therapeutic drugs development of anti-AD in future. |
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