Regulatory Effect Of Shexiang Wulong Pill On High Permeability Of Vascular Endothelium Induced By Sphingosine-1-Phosphate

Objectives Human umbilical vein endothelial cells(HUVECs)were induced by sphingosine-1-phosphate(S1P)to establish a model of high permeability of vascular endothelium.Observe the regulation effect of Shexiang Wulong Pill extract on the permeability of HUVECs,and preliminary explore the possible mechanism of Shexiang Wulong Pill to protect the vascular barrier and reduce the synovial inflammation of rheumatoid arthritis(RA).Methods HUVECs were divided into three groups,namely control group,S1 P induced group and Shexiang Wulong Pill extract group.1 Transwell method was used to detect the change of HUVECs permeability.2 scratch migration test was used to determine the change of vascular endothelial cell migration.3 Immunofluorescent staining was used to observe the changes of ZO-1,Occludin,and Claudin-5 in the three groups of HUVECs.4 Western Blot was used to detect the expression levels of ZO-1,Occludin and Claudin-5 in three groups of HUVECs cells.Results 1 The permeability experiment results show that the permeability of HUVECs in the S1 P induced group is significantly higher than that in the control group(P<0.01);the permeability of HUVECs in the extract group of Shexiang Wulong Pill was significantly lower than that in the S1 P induced group(P<0.01).2 The scratch test results showed that the migration distance of HUVECs in the S1 P induced group was greater than that in the control group(P<0.01);after cultivating Shexiang Wulong Pill extract,the migration distance of HUVECs cells was significantly reduced compared with the S1 P induced(P<0.01).3 The results of immunofluorescence staining of ZO-1 protein showed that the ZO-1 protein of HUVECs in the control group was mainly distributed on the endothelial cell membrane;the ZO-1 protein of the S1 P induced group showed that the fluorescent staining was weakened and the protein expression was significantly reduced;After intervention of Shexiang Wulong Pill extract,the ZO-1 fluorescence staining was significantly enhanced.Claudin-5 protein immunofluorescence staining results showed that the control group HUVECs Claudin-5 protein was clearly distributed on the cell membrane and cytoplasm,and the cell membrane showed a relatively continuous filamentous ring;In the S1 P induced group,the expression of Claudin-5 connexin in the cell membrane was weakened;after intervention of Shexiang Wulong Pill extract,the expression of Claudin-5 was significantly up-regulated.Occludin protein immunofluorescence staining results showed that the Occludin protein of HUVECs in the control group was obviously distributed at the cell edge,and the fluorescence staining was obvious;the Occludin fluorescence staining at the cell edge was obviously missing in the S1 P induced group;after the intervention of Shexiang Wulong Pill extract,the expression of Occludin was significantly up-regulated.4 Western Blot results showed that compared with the control group,the protein expressions of ZO-1,Occludin,and Claudin-5 in HUVECs of the S1 P induced group was significantly reduced(P<0.01).Compared with the S1 P induced group,the expression of all three proteins in Shexiang Wulong Pill extract group increased(P<0.05).Conclusions The extract of Shexiang Wulong Pill can up-regulate the expression of ZO-1,Claudin-5 and Occludin connexin,which can obviously inhibit the increase of the permeability of HUVECs induced by high concentration of S1 P.It is revealed from the molecular level that Shexiang Wulong Pill inhibits RA Possible mechanism of synovial inflammation.Figure 6;Table 2;Reference 165...