| The couplet medicines of rhizoma anemarrhenae and cortex phellodendri was first derived from?Lan Shi Mi Cang?which was written by Li gao in Jin Dynasty.It was used as a classical form of drug suit that always named as "clearance pill" or"kidney pill".It was reflected in many classical prescriptions,such as "Kidney Enriching Gate-Opening Pills”and“zhibai dihuang pills " and so on.In this drug suit,rhizoma anemarrhenae is the dried rhizome which was came from Anemarrhena asphodeloides Bge.of liliaceous plant,it has the effects of clearing heat and purging fire and Nourishing yin and moistening dryness.cortex phellodendri is the dried bark which was came from Phellodendron Chinese Schneid.of Rutaceae plants,it has the effects of heat-clearing and damp-drying,purging fire for removing toxin.The two drugs was always used together and have the efficacy to promote and complementary to each other,Enhancing the efficacy of nourishing yin to reduce pathogenic fire.Modern pharmacological research shows that the chemical composition such as Saponins,flavonoids and polysaccharides in rhizoma anemarrhenae have Hypoglycemic,lipid-lowering and anti-inflammatory effects;and the main effective material for total alkaloids of phellodendron with berberine,has obvious effects of hypoglycemic,lipid-lowering,anti-inflammatory and immune regulation,promote antibody production in mice role.This subject is based on the research status at home and abroad and previous experiments experience,made the effective parts of the couplet medicines of rhizoma anemarrhenae and cortex phellodendri(mainly for total saponins,total alkaloids)as the research object,to explore the mechanism of hypoglycemic and cognitive impairment in insulin dependent signaling pathway(IRS-1/PI3K/AKT),In order to further develop it as a safe,effective and controllable drug for lowering blood sugar and improving cognitive impairment of diabetes.Methods:80 male SPF SD rats,divided into 8 groups with 10 for eatch group,named as normal control group(NC),Alzheimer's disease group(AD),Diabetes mellitus model group(DM),Complex model group(AM),Memantine Hydrochloride administration of AM group,Metformin Hydrochloride administration of AM group;High/Low dose of effective parts of drug couple of Anemarrhena and Cotex phellodendri administration of AM group.Intraperitoneal injection of STZ and high fat feed to induce type 2 diabetes models,gavaging AlCl3 induced dementia model and combination of the two modeling methods to induce complex model.Continuously gavage the rats with Anemarrhena effective part for 20 weeks,determinate the serum GLU?TC?TG?INS?A?1-42 and other beheaviors indicators in 8 and 16 week respectively,analyzes the changes and the relationship between type II diabetes and cognitive impairment.At the end of 20 weeks of administration,dissecting and preservatiing the organ tissues of rat,and the hippocampus and pancreas to made into routine pathological sections for pathology morphology inspection.By Fluorescence quantitative PCR method ang Western blotting method detect the phosphatidyl inositol(3)kinase(PI3K),protein kinase B(PKB is also called AKT protein,Bcl-2 protein expression of brain tissue.Results:1.3 weeks later after Injection of STZ,compared with the normal group,the weight of the model group increase significantly,and FPG significantly increased(P<0.05),indicating that type II diabetes building success.2.Administration 8 and 16 weeks,FPG,TC and TG level in rats:compared with normal group,blood glucose of DM and AM model group rats increase significantly,have the meaning of meter(P<0.01 or P<0.05);Compared with DM and AM model group,the Anemarrhena effective parts has better reduce FPG,TC and TG level(P<0.01 or P<0.05);And the FPG of AD model group has no significant difference,but the levels of TC,TG showed significant differences(P<0.01 or P<0.05).3.For 8 weeks,16 weeks,the results of Barnes maze and Morris water maze experiment showed that,compared with the control group,the time of the 3 model group to find the target hole or the target area latency were statistically significant increased(P<0.001 or P<0.01 or P<0.05),shows that with the modeling time,the rats of model group gradually show cognitive,behavior and memory disorders;compared with the model group,the memantine group and the high dose of effective parts of drug couple of Anemarrhena and Cotex phellodendri administration of AM group show significant effect(P<0.01 or P<0.05),and showed a certain dose effect relationship.4.For 8 weeks,16 weeks of A beta 1-42 protein level results showed that with increasing modeling and administration period,compared with the control group,the model group showed significant differences(P<0.01),suggesting that A beta 1-42 protein level is a gradual accumulation process;compared with the model group,memantine hydrochloride group and Drugs on the high dosage group of A beta 1.42 protein level has improved(P<0.05).5.Administration of 20 weeks INS levels compared with the control group,the model group of peripheral insulin levels and central insulin levels were significantly increased(P<0.001 or P<0.01 or P<0.05);compared with the model group,drug group effective parts of drug couple of Anemarrhena and Cotex phellodendri on improving insulin level is better than the positive group.6.Pancreatic tissue HE staining showed:the control group of pancreatic acinar cells were encapsulated,islet group is round or oval,clear nucleus,clear boundary,each mass of pancreatic islet cells quantity,abundant cytoplasm;3 model groups islet cell disorder,islet cell necrosis,inflammatory cell infiltration and focal islet cell edema,cell ballooning,part of hemosiderin deposition,part of intralobular duct protein tube;effective parts for Anemarrhena pancreatic cells treated with mild edema,compared with model group,cells arranged orderly,pancreatic necrosis loss,small cell ballooning,the relative model group with repair.7.Hippocampus CAl HE staining showed that the neurons of control group rats arranged orderly,cell volume increased,regular shape,uniform color,large round nuclei and the number of Nissl bodies;the number of neurons in hippocampus of the 3 model group significantly decreased,a large number of nuclear pyknosis Nissl body,reduce the number of visible pyknosis the necrosis of neurons,microglia increased,neuronal cell disorder,the effective part of Anemarrhena and Cotex phellodendri delivery quantity of Nissl's group of hippocampal neurons decreased,the number of missing is lighter than the model groups,a small amount of pyknotic necrotic neurons.8.The levels of PI3KmRNA,AKTmRNA,Bcl-2mRNA in the brain tissue:compared with control group rats,PI3KmRNA and AKTmRNA expression in model group rats showed a downward trend,while the Bcl-2 mRNA expression was down regulated(P<0.05);compared with the model group,memantine hydrochloride group and high dose of drug couple of Anemarrhena and Cotex phellodendri group up-regulated the expression of PI3KmRNA,Bcl-2 mRNA and AKTmRNA significantly(P<0.01 or P<0.05);The expression ofthe PI3K Zhibai low dose group rats,AKT mRNA expression is not obvious,can up regulate the expression of Bcl-2 mRNA(P<0.05),showed that the effective fractions of Zhibai drug can effectively inhibit cell apoptosis,repair the nerve injury,possibly through activation of PI3K/AKT pathway.9.Expression of IRS-1,PI3K,AKT,Bcl-2 and Bax protein in brain tissue showed:compared with the control group,AD and AM model group,the protein expression of IRS-1 and AKT was significantly down regulated(P<0.001 or P<0.01 or P<0.05),and the expression of PI3K and Bcl-2 in the model group was significantly lower(P<0.001 or P<0.01);the pro apoptotic Bax protein expression increased significantly(P<0.01);the protein expression in DM model group generally increased(P<0.001 or P<0.01 or P<0.05);compared with the model group,the medicine of the effective fractions of high Zhidai group of IRS-1,PI3K and Bcl-2 protein expression were significantly up-regulated(P<0.001 or P<0.01 or P<0.05);the expression of Bax protein were significantly lower(P<0.001 or P<0.01),suggesting that the administration of drugs on Zhibai effective fractions,can effectively inhibit the apoptosis of nerve cells in brain tissue in a dose-dependent manner.Conclusion:l.The effective parts of Anemarrhena and Cotex phellodendri showed significant effect of regulating glucose and lipid metabolism,as well as improving cognitive impairment of the three kind of models,intraperitoneal injection of STZ and high fat feed to induce type 2 diabetes models,gavaging AlCl3 induced dementia model and combination of the two modeling methods to induce complex model.2.The effective parts of rhizoma anemarrhenae and cortex phellodendri can significantly reduce the A group-beta 1-42 protein accumulation,protein level expression of PI3K and AKT,enhance the expression of Bcl-2 protein,inhibiting the expression of Pro apoptotic gene Bax protein on hippocampus neuron and pancreatic cell injury has repair and protection,and the combination of fluorescence quantitative PCR and WB experimental results show.The changing tendency of mRNA and the expression of the corresponding protein is basically the same.Therefore,we speculated that the drug effect of Zhibai rat nervous system IRS-1/PI3K/AKT/Bcl-2 signal system of effective fractions can lead to diabetes,rat neural apoptosis and cognitive impairment effect. |
PDF Full Text Request