| Purpose: In this paper,the ultrastructure of hippocampal,hypothalamus and small intestine of rats with spleen Yang deficiency syndrome,the changes of signal pathways related to energy metabolism and the therapeutic effect of fu zi li zhong pill were observed by using behavioral and molecular biology methods.The aim is to provide experimental basis for elucidating the biological mechanism of spleen deficiency syndrome and the scientific connotation of the compound’s effect of the “warming middle invigorating spleenin”.Material and method:1.Experimental animal group:60 SPF male Wistar rats,only 7-8 weeks,weight of 200±10 g,adaptive breeding a week later,stratified according to body mass,by using random Numbers were random Ly divided into 4 groups: blank control group(control group),spleen Yang deficiency model group(model group),spleen Yang deficiency + Fu zi li zhong pill group(Chinese traditional medicine group),spleen Yang deficiency natural recovery group(natural recovery group),15 rats in each group.2.Animal model establishment: the rats of control group not exert any interference factors.Composite factors intervention spleen Yang deficiency animal model is set up on the rats of model group.The rats of Chinese traditional medicine group and natural recovery group in copying animal model according to the model group methods.3.Animal model evaluation:Draw up the spleen Yang deficiency syndrome model rat human macroscopic surface syndrome.The main symptoms including: mental fatigue(body mass degradation);lack of power(decreased motor ability and behavioral changes);weak(decreased foreleg holding power);hypothermia;diarrhea(fecal moisture content increased).The minor symptoms including: food intake reduction;fur without burnish.Evaluation criteria: on the 28 th day of animal model replication,through the comparison the changes of body mass,anal temperature,food intake,fecal moisture content,motor ability,behavior and the forearm tension in the rats.The success of animal model replication was objectived evaluation by comparison of differences between groups and the experimental animals that did not meet the evaluation criteria were excluded.4.Drug intervention: after the model establishment,the rats of Chinese traditional medicine group was intragastric gavage with fu zi li zhong pill which was dissolved in physiological saline according to the proportion of 1:1,dosage of 2 m L/100 g·day,for 14 days.AT the same time,the rats of blank group and natural recovery group was intragastric gavage with37℃ saline lavage.Meanwhile,the rats of model group continued to apply the intervention factors of spleen Yang deficiency modeling.Rats in each group were sacrificed on the 43 rd day and animal tissue samples were collected5.The detection of related biological signs and symptoms: respectively after the end of the model establishment and drug intervention,to observe the general condition of rats,anus temperature,measurement of body quality and food intake in metabolic cages testing 24 hours,fecal moisture content.6.Behavioral detection: respectively after the end of the model establishment and drug intervention,using the open field test system(Cheng Du Tai Meng TM-Vision Behavioral detection system)to detect the rat behavior performance,analysis the change of activity indicators,such as movement time,movement distance average velocity(movement distance/time),active area.7.Foreleg holding power measurement: the changes of rats’ forelimb power were tested by using the holding power meter(YLS-13A).8.By using Electron microscope to observe the changes of the mitochondria μLtra structure in intestinal epithelial cells,neurons of Hippocampus and hypothalamus tissues.9.Radioimmunoassay method to detect content changes of ATP in the intestine.10.ELISA method to detect the intestine,hippocampus,hypothalamus tissues beta EP,cAMP and PKA content changes.11.Immunohistochemical SP method to detect the expression changes of beta-endorphin mu receptor in the intestine,hippocampus,hypothalamus tissues.12.RT-PCR method for detection changes of CREB,Sirt1 and mitochondrial UCP2 mRNA expression in intestinal epithelium cells.13.To observe the expression changes of CREB,Sirt1 and mitochondrial UCP2 in intestinal epithelium cells by using Western blotting method.14.The changes of ROS content in hippocampus and hypothalamus were detected by chemical fluorescence.15.Statistical analysis: all data adopt (?)+S,application SPSS19.0 statistical software for data analysis.The difference test was analyzed by single factor variance analysis or rank sum test.Results:1.Compared with the blank group,the body mass,food intake,anal temperature of model group rats decreased obviously(P<0.01),feces moisture content increased significantly(P<0.01),horizontal motion,upright times,movement time,average speed and the fore tension significantly lower than the blank group(P < 0.01);Compared with model group,Chinese medicine group rats presents a significant increase in body mass and food intake(P<0.01),fecal moisture content is reduced(P<0.01),horizontal motion,upright times limbs,average velocity and tension is obvious higher than that of model group(P < 0.01),spleen Yang deficiency syndrome humanoid macro representation improved significantly;Natural recovery group rats have changes similar to those of model group rats.。2.Compared with the blank group,the intestinal epithelium cells,hippocampus and hypothalamus neurons of model group rats μLtra structural damage obviously,mitochondria decrease in the number,swell and fracture,,mitochondria cristae swell and cavity;Compared with model group,the intestinal epithelium cells,hippocampus and hypothalamus neuronsμLtra structural damage degree of Chinese medicine group rats are reduced;Natural recovery group rats have similar changes of μLtra structural to those of model group rats in the intestinal epithelial cells,hippocampus and hypothalamus neurons.3.Compared with the blank group,ATP content in the intestine tissues of model group rats decreased significantly(P<0.01);Compared with model group,ATP content in the intestine tissues of Chinese medicine group rats increased(P<0.05 or P<0.01);ATP content in the intestine of natural recovery group rats are similar to those of model group.4.Compared with the blank group,beta-EP content and mu receptor expression in the intestine,the hippocampus and hypothalamus tissues of model group rats increased significantly(P < 0.01);Compared with model group,beta-EP content and mu receptor expression in the intestine,the hippocampus and hypothalamus tissues of Chinese medicine group rats decreased(P<0.05 or P<0.01);Beta-EP content and mu receptor expression in the intestine epithelial cells,the hippocampus and hypothalamus neurons of natural recovery group rats are similar to those of model group.5.Compared with the blank group,the cAMP and PKA content in the intestine,the hippocampus and hypothalamus tissues of model group rats decreased obviously(P <0.01);Compared with model group,the cAMP and PKA content in the intestine,the hippocampus and hypothalamus tissues of Chinese medicine group rats increased(P<0.05 or P < 0.01);In the intestine,the hippocampus and hypothalamus tissues of natural recovery group rats,cAMP and PKA content lower than that of traditional Chinese medicine group rats(P<0.05 or P<0.01).6.Compared with the blank group,the ROS content in the hippocampus and hypothalamus tissues of model group,Chinese traditional medicine group,natural recovery group rats increased obviously(P < 0.01);Compared with model group,the ROS content in the hippocampus and hypothalamus tissues of Chinese medicine group rats decreased obviously(P<0.01);In the hippocampus and hypothalamus tissues of natural recovery group rats,ROS content significantly higher than that of traditional Chinese medicine group rats(P<0.01).7.Compared with the blank group,the CREB,Sirt1 mRNA expression in intestinal epithelial of model group rats significantly decreased(P < 0.01),mitochondrial UCP2 mRNA expression increased significantly(P<0.01);Compared with model group,the CREB,Sirt1 mRNA expression in intestine epithelium of Chinese medicine group rats increased(P<0.01),mitochondria UCP2 mRNA expression significantly reduced(P < 0.01);Compared with Chinese traditional medicine group,the CREB,Sirt1 mRNA expression in intestine epithelium of natural recovery group rats decreased(P < 0.05 or P < 0.01),mitochondrial UCP2 mRNA expression increased(P<0.05).8.Compared with the blank group,the CREB,Sirt1 protein expression in intestinal epithelium of model group rats significantly decreased(P < 0.01),mitochondrial UCP2 expression increased significantly(P<0.01);Compared with model group,the CREB,Sirt1 protein expression in the intestine epithelium mucosa cells of Chinese medicine group rats increased(P < 0.05),mitochondria UCP2 expression significantly reduced(P <0.01);Compared with Chinese traditional medicine group,the CREB,Sirt1 protein expression in intestinal epithelial of natural recovery group rats decreased(P < 0.05 or P < 0.01),mitochondrial UCP2 expression increased(P<0.05).Conclusion:1.It is showed obvious damage to the ultrastructure of hippocampal,hypothalamic neurons and intestinal mucosal epithelial cells in the spleen Yang deficiency model rat,the number of mitochondria decreased,mitochondrial ridge swelling,fracture,vacuolation,etc.Fu zi li zhong pill can reverse its damage to different degrees.2.In the rats of spleen Yang deficiency model,the content of beta-endorphin and the expression of mu receptors in hippocampal,hypothalamus neurons and small intestinal mucosa epithelial cells were significantly up-regulated,and the activity of cAMP-PKA signaling pathway was inhibited,and fu zi li zhong pill can reverse this abnormal changes to varying degrees.3.The expression of CREB and Sirt1 genes and proteins in the intestinal mucosal epithelial cells of rats with spleen Yang deficiency was significantly down-regulated,while the expression of mitochondrial UCP2 was up-regulated.Fuzi lizhong pill can reverse it.4.The content of ROS in the hippocampus and hypothalamus of rats with spleen Yang deficiency was significantly increased and ATP in the small intestine was decreased.Fu zi li zhong pill can reduce ROS in hippocampus and hypothalamus and increase ATP content in small intestine.5.The fu zi li zhong pill of action of “warming middle invigorating spleenin”aconite may be involved in the ultrastructure protection of central neurons,improve the activity of the beta EP-cAMP-PKA signaling pathway,and promote the energy metabolism of small intestinal mitochondria. |
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