| The construction and optimization of the spleen-deficiency animal model is an important part of the study of spleen-qi deficiency syndrome,and the evaluation criteria of the model is the key link for the successful construction of the spleen-qi animal model.At present,the evaluation of the appearance and signs of the spleen-deficiency animal model is mainly based on qualitative description,lacking accurate and objective quantitative judgment criteria,and it is easy to cause deviations in the evaluation of the appearance of the model animal.Therefore,it is necessary to construct an animal model of spleen-qi deficiency that meets the requirements of scientific research.method-Salivary alpha-amylase(sAA)activity ratio before and after acid load,urine D-xylose excretion rate is an objective reference index for the evaluation of the efficacy of spleen deficiency syndrome recognized by the industry.Studies have shown that sAA belongs to N-glycosylation protein and its activity is closely related to N-glycosylation modification;glycosyltransferases(GnT-Ⅰ and GnT-Ⅱ)and α-mannosidase Ⅱ(MG-Ⅱ)are The key enzyme of N-glycosylation modification directly affects the degree of N-glycosylation.Urinary D-xylose excretion rate,sAA activity ratio and its influencing factors(such as N-glycosylation modification)can be used as an important objective index to evaluate the rat model of spleen qi deficiency."Evaluation by prescription" is a common method for evaluating syndrome models.Yiqi Jianpi represents prescription Sijunzitang is often used to intervene in spleen-qi deficiency model to evaluate the reliability of spleen-deficiency animal model.ObjectiveBased on the appearance and physical signs of spleen-qi deficiency syndrome,the evaluation criteria of rat model of spleen-qi deficiency were optimized;the optimal dose of rats with spleen-qi deficiency caused by reserpine powder was screened,and the optimized animal model of spleen-qi deficiency syndrome was established;on this basis,the spleen-qi deficiency syndrome was obtained.Objective Diagnostic index of urinary D-xylose excretion rate,sAA activity change and its influencing factors to evaluate the rat model of spleen-qi deficiency caused by reserpine;observe the effect of Si junzi decoction on sAA activity and its influencing mechanism in rat model of spleen-deficiency induced by reserpine The impact;to provide a reference for the discussion of the "micro-indicators of spleen-qi deficiency syndrome"and the meaning of "spleen master".Methods1.Optimizing the evaluation criteria of appearance and physical signs of spleen-deficiency rats:selecting activity degree,eye-split width,hair color and stool to form the appearance and physical condition score R of spleen-deficiency rats,as semi-quantitative evaluation standard of appearance signs;subcutaneous injection of reserpine in rat neck Modeling at different doses(0.8,0.4,0.2 mg·kg-1),preliminarily establishing the feasibility of evaluation criteria for appearance signs;replicating the model with 0.8 mg·kg-1 reserpine,and increasing blood glucose by 0.6 mg·kg-1 The flat dose group modeling experiment was used to repeatedly verify the objectivity and reliability of the appearance and physical evaluation criteria to establish a experimental model of the spleen qi deficiency model induced by reserpine,and to select the appropriate dose of reserpine for subsequent spleen deficiency.Mouse model.2.Determination of urinary D-xylose excretion rate by p-bromoaniline method.3.The sAA activity was determined by the Bernfeld method.4.Lectin affinity precipitation method to detect the sAA N-glycan chain composition ratio.5.Western blot analysis was used to detect the expression of sAA,GnT-Ⅰ,GnT-Ⅱ and MG-Ⅱ proteins.6、The histopathological observation of the parotid gland:the morphology of the parotid gland was observed by HE staining.The ultrastructure of the parotid gland(the change of organelles and the changes of zymogen secretory granules before and after acid stimulation)were observed under transmission electron microscope.7.Extraction and analysis of water extract of Si junzi soup:Weighed four herbs(Ginseng,Poria,Atractylodes,and licorice),soaked,decoction,concentrated and freeze-dried to obtain Sijunzi Decoction,high performance liquid phase detection Four gentlemen’s soup water extract map.8.Method and observation of Si junzi decoction:Rats were randomly divided into normal control animals and model animals;model animals were injected subcutaneously with reserpine 0.6 mg·kg-1 for 11 days.At the end of the injection,the model rats were randomly divided into four groups:high-dose and low-dose groups(10g·kg-1,5g·kg-1),fluoxetine hydrochloride(abbreviated as Baiyoujie,3.5 mg·kg-1).Ginsenoside Rbl(40mg·kg-1)group,model recovery group;Si junzi decoction group(10g·kg-1,5g··kg-1),fluoxetine hydrochloride(Baiyoujie)group,rats The rats in the ginsenoside Rbl group were intraperitoneally injected,and the model recovery group was intragastrically administered with the same amount of normal saline(model 11d+22d)for 33 days.After the experiment,the rats were collected saliva and urine,and the rats were selected.Parotid tissue was used for subsequent experiments.Results一、Optimization of Reserpine-induced Spleen-qi Deficiency Rat Model1.Optimization of Appearance Signature Scoring Criteria:According to the appearance Signature Scoring Criteria of rats,the general state change process of model rats was monitored every day during the experiment.The spleen qi deficiency model was successfully replicated or restored to normal nodes with the physical score of R>8 or R<1.The model was evaluated by the appearance sign score combined with body weight,food intake,drinking water,rectal temperature and parotid gland tissue morphology examination,and the model evaluation criteria were optimized;The results were repeated in 9 days after reconstitution of 0.8 mg·kg-1 of reserpine,and were also confirmed in 11 days of reserpine 0.6 mg·kg-1.2.D-xylose excretion rate in spleen-deficiency rats:The D-xylose excretion rate of model rats was lower than that of normal rats(P<0.05),indicating that the intestinal absorption function of rats with spleen-qi deficiency decreased.3、sAA activity in spleen-deficiency rats:sAA activity in rats before and after acid stimulation was lower than that in normal rats(P<0.01),indicating that salivary secretion was impaired in rats with spleen qi deficiency.4.Observation on the influencing factors of sAA N-glycosylation in spleen-deficiency rats(1)The binding rate of sAA to concanavalin(ConA)and wheat germ agglutinin(WGA):the binding rate of model rats to ConA was higher than that of normal rats(P<0.01);-The structure of the sugar chain changes.(2)Expression of sAA N-glycosylation protein,glycosyltransferase(GnT-Ⅰ,GnT-Ⅱ)and α-mannosidase Ⅱ(MG-Ⅱ)in spleen-deficiency rats:Preliminary experiments showed that the expression of sAA N-glycosylation protein was absent in model rats,and the expression of GnT-Ⅰ and GnT-Ⅱ decreased,while MG-Ⅱ showed no significant change.It is suggested that the loss of sAA N-glycosylation expression and structural changes of sugar chains may be related to the changes of GnT-Ⅰ and GnT-Ⅱ expression-5,Histopathological observation of the parotid gland of spleen-deficiency rats:The HE staining of the parotid gland was observed by light microscopy and ultrastructural observation of transmission electron microscopy showed that the model rats showed obvious pathological changes compared with the normal group.After acid stimulation,the zymogen granules were more than the normal control group,but there was no statistically significant difference.It indicates that the spleen deficiency rats have abnormal tissue morphology.二、Intervention of Sijunzi Decoction on Spleen-Qi Deficiency Rat Model1.Extraction and detection of water extract of Si junzi decoction:The yield of water extract of Si junzi decoction was 34.1%.The sample was detected by high performance liquid chromatography with ginsenoside Re,ginsenoside Rgl,ginsenoside Rbl,ginsenoside Rb2,ginsenoside Re,Glycyrrhizic acid,glycyrrhizin,and citric acid were used as test drugs in this study.2.The effect of Sijunzi Decoction on the appearance and physical signs of rats with spleen-deficiency caused by reserpine:Sijunzi Decoction(10g·kg-1,5g·kg-1)was administered for 22 days,the appearance and physical signs of the test group and the model recovery group were all decreased.The appearance sign of the 10g·kg-1 group of Sijunzi Decoction decreased slightly,but there was no significant difference compared with the model recovery group(P>0.05).3.The effect of Sijunzi Decoction on urinary D-xylose excretion rate in spleen-deficiency rats:There was no significant difference between Sijunzi Decoction(10g·kg-1,5g·kg-1)group and ginsenoside Rbl group and model recovery group.P>0.05);Fluoxetine hydrochloride(Baiyoujie)had higher urinary D-xylose excretion rate than the model recovery group(P<0.01).It is suggested that Sijunzi Decoction and ginsenoside Rbl have no significant effect on the intestinal absorption function of spleen-deficiency rats in this experiment-Fluoxetine hydrochloride(Baiyoujie)can improve the intestinal absorption function of rats with spleen-qi deficiency.4.The effect of Sijunzi Decoction on sAA activity in spleen-deficiency rats:After administration,Sijunzi Decoction(10,5g·kg-1),fluoxetine hydrochloride(Baiyoujie),ginsenoside Rbl group spleen deficiency Rat sAA activity increased,but it was not statistically significant compared with the model recovery group.5.The effect of Sijunzi Decoction on sAA N-glycan in rats with spleen deficiency:After administration,Sijunzi Decoction 10 g·kg-1,5g·kg-1 group,fluoxetine hydrochloride group(baiyou solution),ginseng The sAA and ConA binding rate in the saponin Rbl group was lower than that in the model recovery group(P<0.05).The sAA and WGA binding rate in the 4g·kg-1 group of Sijunzi decoction group was higher than that in the model recovery group(P<0.01).It is suggested that the composition of the active higher sugar chain in the sAA N-sugar chain of Sijunzitang group is more than that of the model recovery group,suggesting that Sijunzi Decoction can improve the abnormality of sAA N-glycan structure in spleen-deficiency rats.6.The effect of Sijunzi Decoction on the histopathology of the parotid gland of rats with spleen qi deficiency:After intervention,the pathological tissues of the parotid gland were observed by light microscopy and transmission electron microscopy,and found that Sijunzi Decoction(10g·kg-1,5g·kg-1)There was no significant difference in pathological and ultrastructural relationship between fluoxetine hydrochloride and Baixuan solution in the normal group.After acid stimulation,Sijunzi decoction 5g·kg-1 and fluoxetine hydrochloride were compared with the model recovery group.The decrease of zymogen granules suggested that the stress function of the drug-administered group was better;it indicated that Sijunzi Decoction and fluoxetine hydrochloride could improve the pathological changes of the parotid gland caused by spleen-qi deficiency rats.ConclusionBased on the appearance and physical sign scores,the rat model of spleen-deficiency caused by reserpine was optimized.The general indicators,objective indicators(urinary D-xylose excretion rate,salivary amylase activity)and sAA related indicators(sAA activity)were observed on the model.,sAA N-glycosylation protein expression,sAA N-glycan structure,glycosyltransferase GnT-Ⅰ and GnT-Ⅱ,a-mannosidase MG-Ⅱ,etc.;Sijunzi Decoction on sAA activity in spleen-deficiency rats The changes of its influencing factors have certain intervention effects.The research results provide an optimized evaluation method for the rat model of spleen-qi deficiency caused by reserpine,which provides a research basis for exploring the molecular mechanism of "spleen controlling saliva". |
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