| Objective: To reveal the molecular mechanism of regulating PI3K/Akt/ mTOR signaling pathway by Jianpi yiqi recipe and intervening the process of mesothelial cells' mesenchymal transition.To explore the biological nature of the "deficiency of spleen qi " and the close relationship with the transdifferentiation of peritoneal mesothelial cells from the molecular level.Methods: A total of 75 SD male rats were randomly divided into 5 groups,weight range in 160g-200 g,and 15 rats in each group.After one week of establishment of uremic rat model,peritoneal dialysis was performed immediately except sham operation group,and combined with LPS high glucose induction intraperitoneal injection.(1)The Jianpiyiqi group(high dose group and low dose of Chinese medicine group): to Jianpiyiqi particle flushing by morning gavage.(2)The Atorvastatin group:put the tablets grinding into powder,using the effective dose of 1.8mg/(kg-D)at the start day of peritoneal dialysis.(3)The model group: in the beginning of the rat peritoneal dialysis day with equal volume of distilled water by gavage once every morning.(4)The sham operation group: no 5/6 nephrectomy rats,normal feeding,do not do any intervention.After 30 days,the inner peritoneum tissue of each rat was taken,the size of 1cm x 1cm was measured,and then observe the morphological changes of the peritoneal tissue.Through the tail vein blood testing method to determine the uremic rat model established successfully or not,and by HE staining to observe the thickness of peritoneum and degree of peritoneal fibrosis,morphological changes were observed under the electron microscope,and use the immunofluorescence and Western blot methods determine the expression of alpha-SMA,E-Cadherin,PI3 K,p-mTOR and p AKT proteins.Results:(1): A.light microscope Pathology under light microscope after HE staining of the peritoneal tissue sections were observed in sham operation group by peritoneal cells of normal flat package,a continuous distribution of mesothelial cell structure is complete,and there is a thin connective tissue attached to the peritoneal mesothelial below,no connective tissue hyperplasia.The peritoneal tissues of model group and atorvastatin group,Jianpiyiqi Fang group were thicker than those of sham operated control group,and the texture was loose,accompanied by the loss of mesothelial cells.The mesothelial cells and fibrosis tissues in the model group were blurred,and there was a lot of fibrin deposition in the subcutaneous tissue,which was more obvious than that in the western medicine and Chinese medicine intervention group.B.electron microscopy showed that the peritoneal mesothelial cells in the sham operation group did not deform,and the structure was intact without shrinking.Intracellular organelles and other substances were clearly visible,and mesothelial cells were connected more closely.Compared with the sham operation group,the number of peritoneal mesothelial cells in the model group decreased,most of them had been shedding and deforming,and some organelles in cells had been swollen and deformed,and their original structures were damaged,and fibrous tissue hyperplasia occurred.In western medicine group,cell structure was complete than that in model group.There was a certain accumulation of matrix between mesothelial cells and obvious connective tissue hyperplasia.In the low dose group,the mesothelial cells were observed to fall off,part of the cells were fused with the extracellular matrix,the villi of the cells were occasionally seen,and the intercellular organs were not distinct.In the high-dose group,the fibrosis degree of peritoneum was slight,and the intercellular connection was close.The matrix hyperplasia was not obvious.The mitochondria and endoplasmic reticulum were distributed more clearly,and extracellular fibrin deposition was less.(2)Immunohistochemistry: expression of A.alpha-SMA,the E-Cadherin in the sham operation group and normal expression of alpha-SMA,E-Cadherin was highly expressed in the model group;alpha-SMA showed high expression,the expression of E-Cadherin is low(P<0.05).The expression of alpha-SMA in atorvastatin western medicine group,low dose Chinese medicine group and high dose Chinese medicine group was lower than that in the model group.On the contrary,the expression of E-Cadherin increased compared with the model group(P<0.05).The expression of alpha-SMA in high dose group was lower than that in low dose group and Western medicine group(P<0.05).The expression of E-Cadherin in high dose group was higher than that in low dose group and Western medicine group(P<0.05).B.The expression of B.PI3 K,p AKT and pmTOR was lower in sham operated group than in sham operation group.The expression of PI3 K,p AKT and pmTOR in model group increased significantly(P<0.05).The expressions of PI3 K,p AKT and pmTOR in atorvastatin western medicine group,low dose Chinese medicine group and high dose Chinese medicine group were significantly lower than those in the model group(P<0.05).The expression of PI3 K,p AKT and pmTOR in high dose group of traditional Chinese medicine was lower than that of low dose group and Western medicine group(P<0.05).(3)Western blot: the model group of p AKT and the expression of pmTOR was significantly higher than the sham group;and atorvastatin group,Chinese medicine high dose group and low dose of Chinese medicine group in p AKT and pmTOR expression was significantly reduced compared with the model group;the p AKT and pmTOR in the high dose of Chinese medicine group was obvious decreased than western medicine group and low dose of Chinese medicine group(P<0.05).Conclusion(s):(1)It has been proved by experiments that activation and upregulation of PI3K/Akt/mTOR signaling pathway is one of the main mechanisms involved in mesothelial cell's EMT and peritoneal fibrosis.(2)Jianpiyiqi decoction could upregulate the expression of E-cadherin,inhibit the expression of alpha-SMA and PI3K/Akt/mTOR signaling pathway,thereby inhibiting peritoneal mesothelial cells in the EMT process,and also can reduce the degree of peritoneal thickening and improve peritoneal function,finally to prevent peritoneal fibrosis. |
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