| Objective: The experiment aims to explore the mechanism of action of Qutanhuoxue granules regulating the expression of AQP9 on the cell and molecular level,through observing the expression of AQP9 and P38MAPK signaling pathway in primary cultured rat hepatocytes treated by Qutanhuoxue granules containing serum.Methods : 1?After being fed for 1week,20 male SD rats were randomly divided into blank serum group and medicated serum group.Blank serum group and the other groups were respectively given with normal saline(2ml/d)and Qutanhuoxue granules(2g/d)by gavage,twice a day in the morning and afternoon for 3 consecutive days.After 1 hour of the last gavage,abdominal aortic blood serum was to be made.2?After being fed for 1week,20 male SD rats were randomly divided into normal group and model group.The normal group and model group were respectively given with normal diet and high fat diet.After being fed for 10 consecutive weeks,observing the pathological changes of liver tissue HE staining and oil red O staining.3?After rats model established,separating rat liver cells of normal rats and NAFLD rats by the improved two steps in situperfusion in vivo digestion method.Typan blue staining cell activity,morphology and properties of the inverted microscope and identified by immunofluorescence of CK-18 cells.4 ? The primary hepatocytes of normal rats as normal group,The primary hepatocytes of NAFLD rats were divided into model group,low dose group,medium dose group,high dose group and P38 MAPK inhibitor group.After cultured 48 h,the normal group and the model group were received 10% blank serum,low dose group was received 7.5% blank serum + 2.5% medicated serum.Medium dose group was received 5% blank serum + 5%medicated serum.The high dose group was treated with 10% medicated serum,P38 MAPK inhibitor group was treated with 10% blank serum+1u M P38 MAPK inhibitor(SB203580)to culture 24 h.Detecting the expression of AQP9 mRNA and P38 MAPK mRNA by Realtime-PCR and detecting the expression level of AQP9,P38 MAPK and P-P38 MAPK protein by Western-blot.Results: 1?HE staining: normal hepatocytes are arranged in neat,and no intracellular lipid vacuoles has been seen.Model hepatocytes are disordered arrangement and cells showed diffuse lipid vacuoles,ballooning degeneration of hepatocytes.Oil red O staining:normal hepatocytes can only be seen a few scattered red lipid droplets and Model hepatocytes showed diffuse red lipid droplets in different sizes and obvious steatosis.2?Each NAFLD rat can be obtained approximately(1-1.5)×108 hepatocytes,the cell survival rate is more than 92% and the cell purity > 98%.After cultured 6h cells were adherent,after cultured 24 hcells were connected as the island,After cultured 48 h cells were connected and integrated like the plate,and the cells were mononuclear,binuclear or polynuclear state.CK-18 immunofluorescence identification is positive.3?Compared with the normal group,the expression of AQP9 mRNA and proteins in model group increased significantly(P <0.05).Compared with the model group,the expression of high dose group and AQP9 inhibitor group mRNA and proteins decreased,the inhibitor group was reduced significantly(P < 0.05),while there is no significant difference between the low dose group and model group(P > 0.05).4?Compared with the normal group,the expression and phosphorylation level of P38 MAPK mRNA in model group increased significantly(P<0.05).Compared with the model group,the expression of P38 MAPK mRNA and proteins phosphorylation in varying degrees in other 4 groups,of which the middle dose group and the inhibitor group was reduced significantly(P < 0.05)and middle dose group and inhibitor group were no significant difference(P > 0.05).Conclusion: 1?Using a modified in situ two step perfusion digestion method can successfully obtain a large number of primary high activity,high purity NAFLD hepatocytes.2?The expression of AQP9 and P38 MAPK in NAFLD rats hepatocytes were significantly higher than those in normal rat liver cells,which indicate that AQP9 and P38 MAPK signaling pathway may be one of the mechanism of action of NAFLD.3?P38MAPK inhibitors can inhibit the expression of P38 MAPK in NAFLD rats hepatocytes.whilethe expression of AQP9 in NAFLD rats hepatocytes decreased significantly,which suggest that P38 MAPK signaling pathway plays a role in the regulation of AQP9.4 ? The Qutanhuoxue granules containingserum can decrease the expression of AQP9 in NAFLD rats hepatocytes and inhibit the expression and phosphorylation of P38 MAPK,which suggest that P38 MAPK signaling pathway may be one of the mechanisms of Qutanhuoxue granule regulating AQP9 expression. |
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