Naofucong Ameliorates Diabetic Cognitive Dysfunction By Inhibition Of Apoptotic Signal Pathway Of Endoplasmic Reticulum Stress In Vivo And In Vitro

[Objective]To investigate neuroprotective effects of Naofucong(NFC),a compound Chinese herbal medicine,on CHOP and JNK apoptotic signaling pathways,and the cross pathway between oxidative stress and mitochondrial damage pathway in experimental diabetic rats and the primary cultured hippocampal neurons under hyperglycemia at the animal,cellular and molecular levels.[Methods]In vivo experiment:The diabetic rat model was established and divided into diabetic model(DM)group,low-dose NFC(NFC-L)group,medium-dose NFC(NFC-M)group,high-dose NFC(NFC-H)group and normal control(Con)group.A blood glucose concentration≥16.7mmol/L indicated a successful model.The rats in low-dose NFC group(0.724g/kg/d),medium-dose NFC group(1.448g/kg/d)and high-dose NFC group(2.896g/kg/d)were administered the medication for 12 weeks,respectively.The rats in control group and diabetic model group were administered same volume of distilled water.Blood glucose was measured before and at 4th,8th,12th week after treatment.After 12 weeks,Morris water maze and Y maze tests were used to measure learning and memory,hippocampal neuron apoptosis was observed by TUNEL assay,and expressions of JNK,CHOP and Bcl2 were detected by Western blot and real-time fluorescence quantitative PCR.In vitro experiment:The primary culture system of newborn rat hippocampal neurons was established.The normal control group(25mmol/L glucose)and the conditioned medium with different concentrations of 50,75,100,125 and 150mmol/L glucose were set.Then MTT method was used to explore the best action time and concentration of high glucose on primary hippocampal neurons.They were divided into normal control(Con)group,high-glucose(HG)group,low-dose NFC(NFC-L)group,medium-dose NFC(NFC-M)group,high-dose NFC(NFC-H)group,CHOP si-RNA intervention group,JNK inhibitor SP600125 group and oxidative stress inhibitor N-acetylcysteine(NAC)group.The expression of CHOP was silenced by si-RNA interference and the transfection efficiency was detected by Western blot.Western blot was used to detect the expression of CHOP,Bcl2 and JNK proteins in hippocampal neurons in the normal control group,the high-glucose group and the NFC intervention group,as well as the protein expression of Bc12 and CHOP in the CHOP si-RNA intervention group.Meanwhile,the expression of JNK protein in the JNK inhibitor SP600125 group and the expression of CHOP and Bc12 proteins in hippocampal neurons in the NAC intervention group were detected.The expressions of CHOP and Bcl2 mRNA in hippocampal neurons in the normal control group,the high-glucose group and the NFC intervention group,and the expression of Bcl2 mRNA in the CHOP si-RNA intervention group were detected by real-time fluorescence quantitative PCR.The expressions of JNK mRNA in the JNK inhibitor SP600125 group and CHOP and Bcl2 mRNA in hippocampal neurons in the NAC intervention group were detected.The apoptosis rate of neurons in each group was determined by TUNEL in situ detection and Annexin V/PI/Hoechst three staining.DCFH-DA fluorescence probe and JC-1 fluorescent probes were used to detect the changes of reactive oxygen reactive(ROS)and mitochondrial membrane potential(MMP)of neurons in each group,respectively.Establishment of a mass spectrometry database for the metabolite of the Chinese herbal compound Naofucong:The high-performance liquid chromatography-high resolution mass spectrometry(HPLC-HRMS)technology was used to establish the metabolites and metabolites spectrum of the the Chinese herbal compound Naofucong Granules and NFC-Containing serum.[Results]Part 1 In vivo experiment:1.Blood glucose:The blood glucose in each group was significantly higher than that in the normal group after the modeling(P<0.01).There was no significant difference in blood glucose between the treatment group and the control group at each time point(P>0.05).2.Morris water maze experiment:Compared with Con group,the latency of DM group was significantly prolonged(P<0.05).The incubation period of NFC-H group was shorter than that of DM group(P<0.05).3.Y maze experiment:compared with Con group,the accuracy of DM group decreased(P>0.05).Compared with the DM group,the accuracy of NFC group was improved,but there was no statistical difference(P>0.05).4.Apoptosis of hippocampal neurons:Compared with the Con group,neurons in the hippocampus of the DM group showed the characteristics of apoptosis.The NFC-H group and NFC-M group had a certain recovery effect on the morphological changes caused by high glucose.5.Expressions of JNK,CHOP and Bcl2 in hippocampus:Compared with the Con group,the protein and mRNA expression of JNK,CHOP in the DM group significantly increased in hippocampus of rats(P<0.001,P<0.01 or P<0.05),and the protein and mRNA expression of Bcl2 significantly decreased(P<0.001,P<0.01).Compared with the DM group,the protein and mRNA expression of JNK,CHOP in the NFC-H group and NFC-M group significantly decreased in hippocampus of rats(P<0.001,P<0.01 or P<0.05)In the NFC-L group,the protein expressions of JNK and CHOP in hippocampal tissue of rats decreased(P<0.01).In NFC-H group,the protein and mRNA expression of Bc12 were up-regulated(P<0.001,P<0.05).The protein level of Bc12 was up-regulated in NFC-M group(P<0.01).Part 2 In vitro experiment:1.Hippocampal neurons were purified by differential adherence,density gradient centrifugation,and addition of cytarabine,etc.,and the hippocampal neurons of SD rats were successfully cultured,and the purity reached 95%after identification.2.Apoptosis rate of hippocampal neurons:Compared with the Con group,the proportion of apoptotic hippocampal neurons in HG group was higher(P<0.01).The proportion of apoptotic neurons in NFC(low-dose,medium-dose and high-dose)groups,CHOP si-RNA intervention group,JNK inhibitor SP600125 group and NAC group were lower(P<0.01).There was no statistical difference in the apoptosis rate of hippocampal neurons among the drug intervention groups(P>0.05).3.Protein and mRNA expression of JNK,CHOP and Bcl2 in hippocampal neurons:Compared with the Con group,JNK mRNA,CHOP mRNA and protein were increased,and Bcl2 mRNA and protein decreased in the HG group(P<0.001,P<0.01 or P<0.05).Compared with HG group,the expressions of JNK and CHOP proteins in NFC-H group,NFC-M group,CHOP si-RNA intervention group,JNK inhibitor SP600125 group and NAC group were significantly decreased(P<0.001).Bcl-2 protein expression was significantly increased in NFC-H group,CHOP si-RNA intervention group,JNK inhibitor SP600125 group and NAC group(P<0.001).Compared with HG group,the expression of JNK mRNA in NFC-H group,NFC-M group,CHOP si-RNA intervention group,NAC group and JNK inhibitor SP600125 group were all significantly decreased(P<0.01).The expression of CHOP mRNA in NFC-H group,CHOP si-RNA intervention group,NAC group and JNK inhibitor SP600125 group were all significantly decreased(P<0.001 or P<0.01).The expression of Bcl2 mRNA in NFC-H,NAC group and CHOP si-RNA intervention group were increased significantly(P<0.05,P<0.01 or P<0.001).4.ROS level of hippocampal neurons:Compared with the Con group,the average fluorescence intensity of ROS in the HG group increased(P<0.001).Compared with HG group,the mean fluorescence intensity of ROS of hippocampal neurons in NFC-H group,NFC-M group,NAC group,JNK inhibitor SP600125 group and CHOP si-RNA intervention group were significantly reduced(P<0.001).There was no significant difference between the NFC-H and NFC-M groups and the NAC group,the JNK inhibitor group and the CHOP si-RNA intervention group(p>0.05).5.The mitochondrial membrane potential(△ψm)of hippocampus neurons:The red/green fluorescence intensity ratio of hippocampal neurons in HG group was significantly lower than that in Con group(P<0.05).Compared with HG group,the red/green fluorescence intensity ratios of hippocampal neurons in NFC-H,NAC,JNK inhibitor SP600125 group and CHOP si-RNA intervention group were significantly increased(P<0.01 or P<0.001).Part 3 Establishment of metabolic substance spectrum database of Chinese herbal compound Naofucong:The high-resolution mass spectrometry was used to analyze the compounds in naofucong granules and naofucong containing serum,and the high-resolution mass spectrometry data were obtained to speculate their molecular composition.In addition,the multi-level mass spectrometry was used to analyze the compounds in naofucong granules and NFC-containing serum.83 compounds were collected in this experiment.At present,29 compounds with possible structure were found through the network database Species.[Conclusion]Part 1 In vivo experiment:1.The incubation period of Morris water maze experiment in DM rats was prolonged after 12 weeks intraperitoneal injection of STZ 60mg/kg,which demonstrated the models of cognitive impairment was successfully established.2.The protein and mRNA levels of JNK,CHOP in the hippocampus of diabetic rats were significantly increased,and the expression level of Bcl2 was significantly decreased,suggesting that the stress response of the central nervous system could be initiated and the cell apoptosis increased.The cognitive decline in diabetic rats may be related to apoptosis induced by endoplasmic reticulum stress response.3.The intervention of traditional Chinese herbal compound Naofucong can significantly improve the above changes and has the effect of improving the cognitive function of diabetic rats.The neuroprotective effect of NFC might be related to the inhibition of endoplasmic reticulum stress and reduction of CHOP and JNK apoptotic signaling pathways.Part 2 In vitro experiment:1.In vitro,high glucose induces apoptosis of hippocampal neurons by activation CHOP and JNK pathway,and the neuronal injury induced by high glucose is also related with the activation of the cross pathway of oxidative stress and mitochondrial damage.2.NFC-containing serum has inhibitory effects on CHOP and JNK apoptosis pathway induced by high glucose,and also decreases the expression of ROS and increases the mitochondrial membrane potential.Part 3 Establishment of a mass spectrometry database for the metabolite of the Chinese herbal compound Naofucong:In this paper,high-performance liquid chromatography-high resolution mass spectrometry(HPLC-HRMS)technology was used to establish the metabolites and metabolites spectrum of the the Chinese herbal compound Naofucong Granules and NFC-Containing Serum.[Innovation]To investigate neuroprotective effects of Naofucong(NFC),a compound Chinese herbal medicine,on CHOP and JNK apoptotic signaling pathways,and the cross pathway between oxidative stress and mitochondrial damage pathway in experimental diabetic rats and the primary cultured hippocampal neurons under hyperglycemia from the aspects of animal,cellular and molecular levels,which provides support for elucidating the important pathogenesis of diabetic cognitive impairment,and also provides a theoretical basis for finding new targets for the treatment of diabetic cognitive impairment.