The Study Of Longzhi Decoction On Effect And Mechanism Of Endoplasmic Reticulum Stress In Human Umbilical Vein Endothelial Cells In Vitro

Objective: To study the effect of Longzhi Decoction(LZD)on the cell activity,migration,tube formation,GRP78,CHOP protein and gene expression in endoplasmic reticulum stress-induced human umbilical vein endothelial cells(HUVEC).Then to explore Longzhi Decoction containing serum on endoplasmic reticulum stress of HUVEC.Methods: The LZD containing serum was prepared according to serum pharmacology on SD rats.HUVECs were cultured in vitro and incubated with corresponding LZD-containing / serum-free and endoplasmic reticulum stress-inducing agent Tunicamycin(TM).(1)The effects of 3 ?g/ ml TM at different induction time(3h,6h,12 h,24h)on the viability of HUVECs were detected by CCK-8 assay.And the effect of different concentrations of TM(0.375 ?g/ ml,0.75 ?g/ ml,1.5 ?g/ ml,3 ?g/ ml and 6 ?g/ ml)on the viability of HUVECs were detected by CCK-8 assay.(2)HUVECs were divided into groups as follows: control group(medium with 10% blank serum),TM group(medium with 10% blank serum,3?g/ml TM),TM + LZD group(medium with 10% LZD-containing serum,3?g / ml TM),Longzhi Decoction group(medium with 10% LZD-containing serum).The effect of each group on the viability of HUVEC cells was tested by CCK-8 assay.The effects of each group on the cell migration of HUVECs were detected by cell scratch migration assay.The effect of each group on the tube formation of HUVECs was examined by cell tube formation assay.Immunofluorescence staining was used to compare the fluorescence intensity of endoplasmic reticulum stress-related proteins GRP78 and CHOP in HUVECs.The protein expression of GRP78 and CHOP in HUVECs in each group was compared by Western blotting.The mRNA relative expression of GRP78 and CHOP in each group of HUVECs was compared by RT-PCR.Results:(1)Compared with the control group,the viability of HUVEC showed a decreasing trend(P <0.05)with the time of TM induction,and the cell inhibition rate reached about 40% at 6h(P <0.05).(2)Compared with the control group,the viability of HUVEC cells decreased with the increase of TM concentration.The cell viability decreased significantly at 1.5 ?g/ ml,3 ?g/ ml and 6 ?g/ ml(P <0.05)The cell inhibition rate was about 40% when the concentration was 3?g/ ml(P <0.05).(3)Compared with the control group,TM inhibited the viability of HUVEC cells significantly(P <0.05).Compared with the TM group,TM + LZD group could significantly increase the viability of HUVECs(P <0.05).(4)Compared with the control group,the migration distance of HUVEC cells in TM group were reduced(P <0.05).Compared with TM group,TM + LZD group could significantly increase the migration distance of HUVEC cells(P <0.05).(5)Compared with the control group,the number of tubular formation of HUVEC cells in TM group was significantly decreased(P <0.05).Compared with TM group,TM + LZD group could significantly promote the tubular formation of HUVEC cells(P <0.05).(6)Compared with the control group,the fluorescence intensity of GRP78 and CHOP in HUVEC cells in TM group were increased(P <0.05).Compared with TM group,TM + LZD group could attenuate the fluorescence of GRP78 and CHOP protein in HUVEC cells intensity(P <0.05).(7)Compared with the control group,the protein relative expression level of GRP78 and CHOP in HUVEC cells in TM group were increased(P <0.05).Compared with the TM group,the protein expression level of GRP78 and CHOP in HUVEC cells in TM + LZD group were reduced(P <0.05).(8)Compared with the control group,the relative expression levels of GRP78 and CHOP mRNA in HUVEC cells in TM group were increased(P <0.05).Compared with the TM group,the relative expression levels of GRP78 and CHOP mRNA in HUVEC cells in TM + LZD group were significantly reduced.Conclusions:(1)The Longzhi Decoction can alleviate the over endoplasmic reticulum stress response induced by tunicamycin in HUVEC cells and enhance the growth activity,cell migration ability and cell tube formation ability of over endoplasmic reticulum stress cells.(2)The down-regulation of endoplasmic reticulum stress-related factors GRP78 and CHOP may be one of the mechanisms by which Longzhi Decoction can reduce excessive ER stress in HUVECs.It may be the dragon leech soup treatment of ischemic brain injury one of the mechanisms.