Intervention Effect And Mechanism Of Yiqi Bushen Recipe On Diabetic Hippocampus Injury

Part One:Evaluation of Effectiveness of Yiqi Bushen Therapy on Cognitive ImpairmentObjective:To evaluate the clinical effect of Yiqi Bushen therapy for cognitive impairment by meta-analysis.Methods:The literatures published in China Biology Medicine Disc(CBM),China National Knowledge Infrastructure(CNKI),WANFANG,VIP,PubMed,SinoMed,Web of science and other databases were retrieved through the Internet.The randomized controlled trials(RCTs)on the Yiqi Bushen therapy of cognitive impairment from the establishment of the database to September 30,2020 wer e searched according to the inclusion criteria,and the literatures meeting the inclusion criteria were screened.Their effectiveness was statistically analyzed and evaluated by Review Manager 5.3.Results:A total of 16 RCTs were included,covering 1216 patients.The results showed that the Yiqi Bushen therapy could significantly improve the clinical efficacy of patients with cognitive impairment[RR=2.2,95%CI(1.52,3.18),z=4.20,P<0.01],as well as their performance in minimentalstate examination(MMSE)[SMD=0.92,95%CI(0.54,1.29),z=4.83,P<0.01]and clock drawing task(CDT)[SMD=0.93,95%Cl(0.54,1.31),z=4.73,P<0.01].Conclusion:Compared with applying conventional western medicine therapy alone,its combination with the Yiqi Bushen therapy has a significantly better performance in improving the clinical efficacy of cognitive impairment and restoring defective neurological function,which is worthy of clinical promotion.However,adverse reactions are not systematically described,and the research quality is low and the sample size is small,so future multi-center RCTs with large number of samples are needed.Part Two:Intervention Effect and Mechanism of Yiqi Bushen Recipe on Diabetic Hippocampus InjuryObjective:To have a deep insight into the effect of Based on the previous clinical experience,the effect of Yiqi Bushen recipe on diabetic hippocampus injury and its internal mechanism,thereby providing basic experimental support for developing the traditional Chinese medicine(TCM)treatment scheme for DM hippocampus injury.Methods:A total of 80 C57BL/6J healthy male mice(weighing 25±2g)aged 8 weeks were randomly included in the normal control group(n=20)and the experimental group(n=60),and then injected intraperitoneally with 1%streptozotocin(STZ)diluted with sodium citrate solution for 5 days.After successful modeling,the experimental group was divided into the DM group,the low-dose TCM group and the high-dose TCM group.The low-dose and highdose TCM groups were intragastrically administered with 200mg/kg/d and 800mg/kg/d Yiqi Bushen TCM suspension,respectively,while the normal control group and the DM group were intragastrically administered with the same amount of double distilled water every day for 12 weeks.During this course,the dynamic changes of body weight and random blood glucose(RBG)of mice were recorded,and their basic conditions of mental state,hair color,diet and urine volume were observed.The behavioral evaluation(Morris water maze)was carried out on mice in each group after 12 weeks of intragastric administration.After the experiment,brain tissue samples and serum were collected.The left brain of mice was soaked and fixed in 4%paraformaldehyde,then embedded in paraffin and sectioned.The morphology of neurons in the hippocampus was detected by hematoxylin-eosin(HE)staining and Nissl’ s staining,and immunohistochemical staining of Krt8 was performed.The hippocampus of the right brain was separated and stored in the refrigerator at-80℃,and the expression level of Krt8 mRNA was detected by qRT-PCR;the content of Krt8 in serum of mice in each group was detected by Elisa.Results:1.General observation:the mice in normal control group had moderate body shape,good mental state,free movement,sensitive response,shiny hair,normal ingestion and water intake as well as normal urine and feces;compared with the normal control group,the mice in the DM group lost weight and had drooped spirit,slow response,and significantly increased ingestion and water intake as well as urine and feces;the mice in the low-dose TCM group and the high-dose TCM group had the same symptoms as those in the DM group,but the symptoms were alleviated.2.Body weight:before modeling,all the mice in each group were healthy mice with normal body weight;after successful STZ modeling,compared with the normal control group,the body weights of mice in the DM group,the lowdose TCM group and the high-dose TCM group were decreased significantly(P<0.01),among which,the body weight of mice in the DM group showed a down trend with fluctuation;after drug intervention,compared with the DM group,the weight of mice in the low-dose TCM group and the high-dose TCM group increased significantly(P<0.05 or P<0.01),but they did not reach the body weight level of mice in the normal contol group(P<0.01).The.re was no significant difference in body weight between the low-dose and high-dose TCM groups(P>0.05).3.Blood glucose:after successful STZ modeling,compared with the normal control group,the blood glucose of mice in the DM group,the low-dose TCM group and the high-dose TCM group was higher than the normal level(>16.7mmol/L),and the mice in the DM group maintained a hyperglycemic state;after drug intervention,compared with the DM group,the blood glucose of mice in the low-dose TCM group and the high-dose TCM group was significantly reduced(P<0.05 or P<0.01),but none of them was reduced to the blood glucose level of the normal control group(P<0.01);after 12 weeks of intragastric administration,the blood glucose level of mice in the high-dose TCM group was significantly lower than that in the low-dose TCM group(P<0.05).4.Behavioral evaluation(Morris water maze)results:Comparison of results during latency:compared with the normal control group,the latency of the DM group was significantly prolonged(P<0.01);the latency of the low-dose and high-dose TCM groups was significantly shorter than that of the DM group(P<0.01),and there was a significant difference between the high-dose TCM group and the low-dose TCM group(P<0.05).Comparison of platform crossing frequency:compared with the normal control group,the platform crossing frequency of mice in the DM group,the low-dose TCM gr oup and the high-dose TCM group was significantly reduced(P<0.01);the platform crossing frequency in the low-dose and high-dose TCM groups was significantly greater than that in the DM group(P<0.05),and there was no significant difference between the low-dose and high-dose TCM groups(P>0.05).5.Hippocampus staining results:HE staining:in the normal control group,the neurons in CA1 region of the hippoca.mpus arranged closely and neatly,with clear layers,large number,large and plump cells,clear nucleoli and intact structure;in the DM group,the neurons in the same region were significantly damaged,with a reduced number,loose arrangement,intercellular space,vague boundary of nuclear and cell membranes,and damaged structure.In the low-dose and high-dose TCM groups,the neurons in the same re gion were mildly damaged and arranged neatly,with clear layers and relatively intact structure.Nissl’s staining:in the normal control group,the neurons in CA1 region of the hippocampus were not damaged,and the pyramidal cells arranged neatly and orderly,with intact structure and clear layer,and Nissl bodies were enriched in the cytoplasm;in the DM group,the neurons in the same region were seriously damaged,and pyramidal cells were disordered,with damaged structure and unclear layers,and Nissl bodies in the cytoplasm were significantly reduced;in the low-dose and high-dose TCM groups,the neurons the same region arranged neatly,with intact structure and clear layers,and Nissl bodies were enriched.6.Immunohistochemical results:in the normal control group,the expression level of Krt8 in CAl region of the hippocampus was low,and there were a small amount of brown-yellow positive granules;compared with the normal control group,the expression level of Krt8 in the DM group and the lowdose and high-dose TCM groups was significantly increased(P<0.05 or P<0.01),and more brown-yellow positive granules were observed;compared with the DM group,the area of Krt8 brown-yellow positive granules in the low-dose and high-dose TCM groups was significantly reduced(P<0.01);and there was no significant difference between the low-dose and high-dose TCM groups(P>0.05).7.qRT-PCR results:compared with the normal control group,the expression level of Krt8 mRNA in the DM group and the low-dose and high-dose TCM groups was significantly increased(P<0.05 or P<0.01);compared with the DM group,the expression level of Krt8 mRNA in the low-dose and high-dose TCM groups was significantly reduced(P<0.05 orP<0.01);and there was significant difference between the low-dose and high-dose TCM groups(P<0.05).8.Elisa results:the content of Krt8 in serum of mice in the DM group and the low-dose and high-dose TCM groups was significantly higher than that in the normal control group(P<0.01);compared with the DM group,the content of Krt8 in serum of mice in the low-dose and high-dose TCM groups was significantly reduced(P<0.05);and there was no significant difference between the low-dose and high-dose TCM groups(P>0.05).Conclusions:1.DM mice modeled by intraperitoneal injection of streptozotocin(STZ)had significantly reduced body weight and significantly increased blood glucose,indicating that Yiqi Bushen recipe could significantly improve the general status,body weight and blood glucose of DM mice.2.The learning and memory ability of DM mice increased significantly,indicating that Yiqi Bushen recipe could significantly improve the learning and memory ability of DM mice.3.Yiqi Bushen recipe can improve the injury of hippocampal neuronal cells in diabetic mice,and thus play a significant role in improving the hippocampus injury of diabetic mice and delaying its progress,which may be related to the down-regulation of the expression of Krt8 in the hippocampus of diabetic mice.