Study On The Mechanism Of Spleen Deficiency In Rats With Spleen - Yin Deficiency Based On RT - PCR And Western Blot

Purpose：Based on the theory of Zang-fu manifestations and further analysis andverification of spleen-yin deficiency rat ileum proteomics differences by RT-PCRand Western blot technology in early period, trying to find and explore spleenyin deficiency syndrome differential protein spectrum to crack the materialbasis and mechanism, combing physiological function of protein and their mutualassociation. Investigating the mechanism of spleen yin deficiency syndrome fromthe view of systems biology, looking for the syndrome specific quantitativeindicators, and tried to construct physiological mechanism of network of spleenyin deficiency, to provide accurate, reliable experimental basis for theclinical diagnosis and treatment of spleen and stomach diseases in later period.Material and method：SPF Sprague-Dawley (SD)36rats,aged3months， were randomly divided intocontrast group，CG, spleen yin deficiency model group and traditional Chinesemedicine disproof group, using the classical compound factors model withassociation of improper diet, overstrain plus consumption of Yin fluid medicine,to establish the spleen yin deficiency rat model. Moldinga total of16days,8days for a stage, the first phase is rat model of spleen qi deficiencysyndrome,adopting methods of improper diet and overstrain.The second phase ismodel of rat with spleen yin deficiency syndrome,based on above intervene, withthe consumption of Yin fluid drug.We evaluate the model by using fuzzymathematical method in eighth days and sixteenth day time node.After theestablishment of model,we give the spleen yin affirmative treatment, rats werefed on standard solid feed in the period of making model, the model group medication treatment and normal group were given the amount of physiologicalsaline. Those in the inclusion criteria were prepared.Ileum of rats were observed by means of Pathology; Judging changes in therelative amounts to mRNA gene transcription level,detecting differences in genelevel in spleen yin deficiency syndrome; Judging target protein changes throughWestern blot,detecting expression differences of related-protein from proteinlevel.Results：1.Ileum pathological observation Spleen yin deficiency rat: The basic structureof ileum wall of healthy control group rats were intact and clear,arrangementrule of mucosal glands is neat without infiltration, necrosis, loss and otherpathological changes. Compared with the model group,multiple edema were foundedin model rats of spleen yin deficiency, the basic structure is notcomplete,intestinal villi become short, shedding. the structure of ilealmucosa of disproof group rats recovered to normal after drug intervention, noobvious damage were observed.2．Results from the expression of mRNA protein detection of RT-PCR method: thesemi uantitative differences of ileum of rats the expression level of mRNAprotein expression analysis found that compared with healthy controls,spleenyin deficiency model group, Canx, CAT inileum tissue ACO2and Papss weresignificantly increased (P<0.05).While the expression of mRNA2protein HSP90protein mRNA was significantly decreased(P<0.01). After the management of spleenyin affirmative intervention, ileum tissue in rats with CAT, Canx, ACO2and Papss2protein expression of mRNA were significantly reduced (P<0.05)compared withthe model group, and HSP90protein expression of mRNA increased significantly(P<0.05), but all the protein compared with the normal group had no significantdifference (P>0.05). β-actin expression had no significant difference in each group (P>0.05)3．Results from the expression of Western blotting protein detection method:semi quantitative differences of ileum of rats analysis of protein expressionlevels found,compared with the healthy control group, spleen yin deficiencymodel group,Canx, CAT in ileum tissue ACO2and Papss2protein were significantlyincreased(P<0.05), among which the most significant (CAT upregulation P<0.01),and HSP90protein content were significantly lowered (P<0.05). Management ofspleen yin affirmative intervention, ileum tissue in rats with CAT, Canx, ACO2and Papss2protein content were significantly reduced(P<0.05)compared withthe model group, and HSP90protein expression of mRNA increasedsignificantly(P<0.05), but all the protein compared with the normal group hadno significant difference (P>0.05). The protein of β-actin expression had nosignificant difference in each group (P>0.05)Conclusion：1spleen yin deficiency spleen affect catalase, the body of the aconitatehydratase, calcium binding protein, Papss gene and protein2and expression ofheat shock protein90, which involved in energy metabolism, tissue, cell signaltransduction, cell proliferation and apoptosis and other aspects of thechange,most of which are closely related to the metabolism sugar, lipid,protein.2spleen yin deficiency may exist as a mitochondrial damage as the foundation,further cell membrane peroxidation injury, Ca2+homeostasis, excesssulfuricacid and protein changes in energy and material pathological-mechanism.