| Objective:(1)To observe the expression of rat colon smooth muscle cells(CSMCs)and colonic tissue brain-gut peptide(LP,NPY and CCK-8),and to explore the correlation between gastrointestinal peptide expression and anorexia;(2)To observe the effect of Li-Pi Compound Preparation drug-containing serum on the regulation of CSMCs proliferation and LP,NPY and CCK-8 in rats,and to explore the mechanism and target of its effect on smooth muscle of colon and treatment of anorexia;(3)To observe the effect of Li-Pi Compound Preparation on the regulation of colonic tissue LP,NPY and CCK-8 in anorexia rats,to explore its therapeutic target and mechanism,and to lay a theoretical foundation for the popularization of the famous Traditional Chinese Medicine Experience Prescription.Methods:The experiment was completed in two parts:1.Effect of different concentrations of Li-Pi Compound Preparation on brain-gut peptide in CSMCs of rats:SPF grade SD rats were 60,30 days old,60±5 each half male and female,of which 10 were used for CSMCs culture and 50 for drug-containing serum preparation.(1)Preparation of serum containing different doses of Li-Pi Compound Preparation:The 50 rats in the serum group were randomly divided into normal group(N group),low dose group(LD group),medium dose group(D group),high dose group(HD group)and zinc sulfate group(Z group),10 rats in each group.Rats in the normal group were given drinking water by intragastric administration,and rats in the other groups were given low,medium and high doses of Li-Pi Compound Preparation and Oral Solution of Zinc Sulfate for 7 days,and to extract pure drug-containing serum from each group of rats.(2)Cultured of CSMCs:Colon tissue was soaked with 2%penicillin streptomycin,and then digested by 0.25%trypsin for 30min.Then colon smooth muscle tissue and its cells were cultured with 25%FBS.Three generations of CSMCs were frozen after immunohistochemical identification.(3)Effects cell proliferation of different concentrations of serum of different doses of Li-Pi Compound Preparation:The pure drug-containing serum of each group was diluted to the concentration of 10%,20%and 30%to intervene the CSMCs,and find out the optimum concentration and time of cell proliferation.(4)The protein concentration,relative protein expression and relative mRNA expression of LP,NPY and CCK-8 in CSMCs were detected after the CSMCs with the best serum concentration of Li-Pi Compound Preparation and zinc sulfate.2.Effects of Li-Pi Compound Preparation on LP,NPY and CCK-8 in the colonic tissues of anorexic rats:Half of the SPF SD female rats and half of male rats,a total of 72 rats,were fed separately.They were randomly divided into the normal group(N group)with 16 rats and the model group(M group)with 56rats.The rats of N group received normal diet,while the M group received special diet of anorexia model.After the success of the anorexia model,6 specimens were collected for the first time to understand the pathological changes of the liver in anorexia rats and so on.In addition to group N,group M was randomly divided into model control group(MC group),low-dose group(LD group),medium-dose group(D group),high-dose group(HD group)and zinc sulfate group(Z group),with 10 rats in each group.Except for drinking water intragastric administration in N group and MC group,the other groups were given low,medium and high doses of Li-Pi Compound Preparation and Zinc Sulfate n.After 7 days of intragastric administration,the colonic tissues of the rats were taken and the concentration of LP,NPY and CCK-8 protein concentration,relative protein expression and relative mRNA expression of the tissues were detected by ELISA,Western Blot and RT-PCR.All experimental data were statistically analyzed using SPSS 21.0 software.Result:Experiment 1 1.The survival rate of the cells cultured by the simplified CSMCs culture method was high,and the positive expression rate of positive positive ?-action was over 95%.2.The optimal concentration of Li-Pi Compound Preparation and zinc of 10%sulfate serum was the serum concentration,and the concentration of LP,NPY and CCK-8 protein,relative expression of protein and relative expression of mRNA in CSMCs of rats could be upregulated after intervention of CSMCs24h.Experiment 2 1.The food intake and body weight of anorexic rats were 63.3%and 24.1%lower than those of normal rats,respectively.The food intake and body weight of anorexic rats continued to increase after the intervention of Li-Pi Compound Preparation and zinc sulfate,but the body weight increased slowly.2.The special feed was divided into high fat,high protein and low carbohydrate,and the anorexia model could be successfully prepared when the rats ingested the feed,and the pathological changes of non-alcoholic fatty liver disease occurred in the liver.3.The expression levels of LP,NPY and CCK-8 protein in the colon tissues of anorexia rats were all lower than that of normal rats(P<0.05),the relative expression levels of protein were all higher than that of normal rats(P<0.05),and the relative expression levels of LPmRNA were higher than that of normal rats(P<0.05).Different dose Li-Pi Compound Preparation on anorexia rats colon tissue LP,NPY,CCK 8 protein concentration were raised(P<0.05),medium dose group by LP protein and mRNA expression quantity relatively lower(P<0.05),low dose group NPY protein relative expression(P<0.05),while the medium and high dose group raised NPYmRNA relative expression(P<0.05),each dose group raised CCK-8 protein and mRNA expression quantity relatively,but protein expression of relative quantity have difference(P<0.05).4.Zinc sulfate up-regulated the concentration of LP,NPY and CCK-8 proteins in the colon tissues of anorexia rats(P<0.05),down-regulated the relative expression of LP and NPY proteins(P<0.05),up-regulated the relative expression of CCK-8 proteins(P<0.05),and down-regulated the relative expression of LPmRNA(P<0.05).Conclusion:1.10%Li-Pi Compound Preparation and zinc sulfate containing medicine serumwere the best concentration of CSMCs proliferation,the enhancement cell activity,may be one of increasing impetus mechanism of colon tissue,and can increase the LP CSMCs,NPY,CCK-8 concentration of protein,protein and mRNA expression quantity relatively,this may be a reason of spleen compound preparations with zinc sulfate one of targets and its mechanism.2.special feed was made of high fat,high protein and low carbohydrate,which is consistent with the pathogenesis of anorexia and the basic principle of TCM etiology simulation.The pathological changes of non-alcoholic fatty liver disease in the liver of rats after ingestion of the feed confirmed that the symptoms of anorexia were closely related to the dietary structure and pathological changes of the liver.3.The concentration of LP,NPY and CCK-8 proteins in the colon tissues of anorexia rats were abnormally decreased,while the relative expression of protein and mRNA was abnormally increased,which may be one of the main mechanisms and targets of anorexia.4.Li-Pi Compound Preparation and zinc sulfate as the control all had regulatory effects on protein concentration,relative protein expression and mRNA expression of LP,NPY and CCK-8 in colon tissues of anorexia rats,which may be one of the main targets and mechanisms of these two drugs in the treatment of anorexia.5.Improved CSMCs simplified culture:After soaking colon tissue with only 2%double resistance,0.25%trypsin was used to digest colon smooth muscle for 30min without oxygen,and 25%FBS was used to jointly cultivate colon smooth muscle tissue and cells,so as to simplify the culture procedure.This simplified culture method of CSMCs has a high cell survival rate and positive expression rate,which is innovative,operable and practical,and worthy of promotion. |
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