Mechanism Research Of Kidney-Yang Deficiency On Digestive And Absorptive Function

Objective: The kidney-Yang deficiency rat model were set by the bilateraladrenal subtotal excision to observe the digestive and absorptive function inmodel rats based on the related theory of spleen and kidney, with spleen yangrooting in kidney-yang as the breakthrough point. Around the serum content ofGAS and D-xylose，the related mechanism was also investigated between theexpression of apoptosis and apoptosis related protein (such as Caspase-3,Bcl-2, Bax) and the digestive and absorptive function effected by kidney-Yangdeficiency. Else, the effect on digestive and absorptive index was observed inkidney-yang deficiency model rat treated by Shenfu Decoction. Modernresearch methods were used to detect the digestive and absorptive indexs,incuding the rate of apoptosis of gastric antrum mucosa tissue and intestinalmucosal absorptive epithelial cell, the expression in gastric antrum mucosatissue’s G cells, the percentage of Caspase-3expressed by G cells, theexpression of Bcl-2and Bax, and ultrastructural pathologic morphology inintestinal mucosal absorptive epithelial cell, et al. Our research aimed toinvestigate the mechanism of digestive and absorptive function effected bykidneyYang deficiency from Molecular gene level.Methods: Male8weeks aged SD rats were adopted，which resected bilateraladrenal subtotal to set up kidney-yang deficiency model, and which withoutresected bilateral adrenal were as sham operation group.4weeks afteroperation, the operative rats were randomly divided into model group andShenfu Decoction group. In every morning8:30, every rat in Shenfu Decoction group was given Suspension liquid of1.6ml/200g ShenfuDecoction through the lavage, and this drug concentrations for each of1mlsolution contained0.5g crude drugs. Every rat in Sham operation group andmodel group both had been given the physiological saline of the same capacityfor4weeks.We determinated the temperature of all experimental rats,collected their blood and24h urine at the end of the experiment. Gastricantrum mucosa tissue and intestinal mucosa tissue were left to test relatedindexes, by separating and collecting intestinal mucosal absorptive epithelialcell:①To detect the content of24hour urinary17-OH-CS;②To detect thecontent of serum GAS and D-xylose;③To observe the microstructurechanges of gastric antrum mucosa tissue and intestinal mucosa tissue;④Toobserve the ultrastructural changes in absorptive epithelial cell of intestinalmucosa tissue;⑤To detect the apoptosis rate of gastric antrum mucosa tissueby TUNEL, the average optical density value of G cells byimmunohistochemistry and the percentage of Caspase-3expressed in G cellsthrough immunohistochemical double staining method;⑥Taking the singlecell suspension of intestinal mucosal tissue absorptive epithelial cells, to detectthe apoptosis rate of absorptive epithelial cell of intestinal mucosa tissue byflow cytometry;⑦To examined the average optical density value of Bcl-2and Bax in intestinal mucosal tissue absorptive epithelial cells byimmunohistochemistry.Results:①Detection of temperature and24hour urinary index: Comparedwith the sham operation group, the temperature and content of urine17-OH-CS of the model group decreased significantly(P<0.01); Comparedwith the model group, the temperature and content of urine17-OH-CS of theShenfu Decoction group increased significantly(P<0.01).②Detection of serum index: Compared with the sham operation group,the content of serum GAS and D-xylose of the model group decreased significantly(P<0.01); Compared with the model group, the content of serumGAS and D-xylose of the Shenfu Decoction group increased significantly(P<0.01).③Detection of gastric antrum mucosa tissue index: The results showedthat of the light microscope：Compared with the sham operation group, theantrum mucosa tissue of the model group became thiner, the cell gap becamelarger. Else, the mucous glands have different degrees of alignment disorders,loose；the emergence of a large number of vacuolar degeneration. Comparedwith the model group, the antrum mucosa tissue of the Shenfu Decoctiongroup was slightly thick, the cell gap became smaller, the mucous glands cellsarranged more regulation, and the vacuolar degeneration had reduced. TUNELand immunohistochemistry results show：Compared with the sham operationgroup, the apoptosis rate of gastric antrum mucosa tissue and the percentage ofCaspase-3expressed in G cells of the model group increased significantly(P<0.01), while the average optical density value of G cells decreasedsignificantly(P<0.01). Compared with the model group, the apoptosis rate ofgastric antrum mucosa tissue and the percentage of Caspase-3expressed in Gcells of the Shenfu Decoction group decreased significantly(P<0.01), whilethe average optical density value of G cells increased significantly(P<0.01).④Detection of intestinal mucosa tissue index: The results showed that ofthe light microscope：Compared with the sham operation group, intestinalmucosa tissue biopsy of the model group showed that mucosaangl thicknesswas less uniform. And the villus edge was irregular, partial shedding of theepithelium fell off and deleted. Compared with the model group, intestinalmucosal thickness of the Shenfu Decoction group was more uniform, thevillus edge was more regular, and intestinal mucosal epithelial tissue was morecomplete. The results showed that of the electron microscope: Compared withthe sham operation group, electron microscopy showed that intestinal epithelial cell microvilli slightly was a little sparse, and junction between cellswas loose. Thus that cell device was fuzzy in cell membrane invaginating. Atthe same time, heterochro-matin of this group had a condensation and frontierset, and cytoplasmic had many vacuoles. However, ultrastructural of intestinalepithelial cells of the Shenfu Decoction group had remarkable improvementcompared with the model group. Compared with the sham operation group,immunohistochemistry result showed that the expression of Bcl-2and Bax inintestinal mucosal absorptive epithelial cell of the model group increasedsignificantly (P<0.01). Compared with the model group, the expression ofBcl-2in intestinal mucosal absorptive epithelial cell of the Shenfu Decoctiongroup increased significantly (P<0.01), while the expression of Bax decreasedsignificantly (P<0.01).⑤Detection of intestinal absorption cell index: Compared with the shamoperation group, the apoptosis rate of intestinal mucosa absorptive epithelialcells of the model group increased significantly (P<0.01). Compared with themodel group, the apoptosis rate of intestinal mucosa absorptive epithelial cellsof the Shenfu Decoction group decreased significantly (P<0.01).Conclusion:①The model rats with deficiency of kidney-yang wasestablished by adrece which conformed to the diagnostic criteria of deficiencyof kidney-yang. Thus the temperature and24h urinary17-OH-CS content ofthe model group rats were significantly decreased. So it suggests that thereplication of this animal model was successful.②Compared with the sham operation group, the content of serum GASand D-xylose of the model group decreased significantly. This result illustratesthat the digestion and absorption function of kidney-yang deficiency animalhad a decline.③The apoptosis rate of gastric antrum mucosa tissue and the percentageof Caspase-3expressed in G cells of the model group increased significantly, while the average optical density value of G cells decreased significantly. So itsuggests that the abnormal apoptosis of gastric antrum mucosa tissue and its Gcell has a influence on secretion of GAS.④Ultrastructural organization structure in intestine absorptive epithelialcells of the model group conformed to the typical features of apoptosis cell.The apoptosis rate of intestinal mucosa absorptive epithelial cells of the modelgroup increased significantly. At the same time, the expression of bax had asignificant increase. Thus there had a remarkable apoptosis in the model rats,and had a unbalance between proliferation and apoptosis. Then the apoptosisof intestinal mucosa absorptive epithelial cell increased. However theproliferation was insufficient which could’t be updated in time. Finally it had anegative effect on absorption function of the model rats.⑤Shenfu Decoction, a prescription for warming and recuperating thekidney-yang, can inhibit the excessive apoptosis of correlated tissue cells byadjusting the expression of Caspas-3、Bcl-2and Bax. Else this drug can alsopromote the cell renewal, maintenance of its function, thereby improving thedigestive and absorptive function of kidney-yang deficiency model rats.Our research first uses the bilateral adrenal subtotal excision method toestablish the kidney-yang deficiency animal model. We take the lead in the useof modern technology and method to study the mechanism of digestive andabsorptive function affected by kidney-yang deficiency.As the key protein ofapoptosis in G-cell of gastric antrum mucosa tissue, Caspase-3expression ofthe kidney-yang deficiency model rats increased remarkably. So did theexpression of Bax which was the pro-apoptotic gene in intestinal mucosalabsorptive epithelial cell. It increased the apoptosis of digestive and absorptiverelated tissue cell in the model rats. Then the performance for the digestiveand absorptive function declined. Shenfu Decoction, a prescription forwarming and recuperating the kidney-yang, can improve the impaired digestive and absorptive function leaded by kidney-yang deficiency throughdecreasing the express of Caspase-3in G cells of gastric antrum mucosa tissue,and Bax in intestinal mucosal absorptive epithelial cell. Thus, abnormalapoptosis may be one of the mechanism that kidney-yang deficiency can havea negative effect on digestive and absorptive function.