Study On Improving Effect And Mechanism Of Bu Shen Huo Xue Prescription On The Cell Model Of Diabetic Cerebral Microangiopathy

Objective:To explore at the cellular level whether the bushenhuoxue prescription has an improvement effect on diabetic cerebral microangiopathy,and to conduct a preliminary study on its mechanismMethods:1.In vitro experiments using high glucose（25mM,HG）and palmitic acid（200μM,PA）intervention mouse brain microvascular endothelial cells（BEND3 cells）to establish diabetic cerebral microvascular lesion cell model.CCK-8 method was used to select the drug concentration of appropriate bushen formula,huoxue formula and bushenhuoxue formula,and to determine whether bushenhuoxue method had any effect on cerebrovascular diseases caused by glucolipotoxicity;2.Elisa was used to detect the effects of bushen formula(BS,25mg·L^-1,50mg·L^-1,100mg·L^-1),huoxue formula(HX,5mg·L^-1,10mg·L^-1,20mg·L^-1)and bushenhuoxue formula(BSHX,30mg·L^-1,60mg·L^-1,120mg·L^-1)on the oxidative stress and inflammatory response related indicators of brain microvessels caused by high glucose and palmitic acid;3.Western blot was used to detect the optimal concentration of bushen formula(100mg·L^-1),huoxue formula(20mg·L^-1)and bushenhuoxue formula(120mg·L^-1)to detect the expressions of glucose regulated protein 78（GRP78）,phospho eukaryotic initiation factor 2α（p-eIF2α）,activating transcriptional factor 4（ATF4）and nuclear factor kappa-B（NF-κB）protein to explore the relationship between diabetic cerebrovascular lesions and endoplasmic reticulum stress;4.Western blot was used to detect the protein expression of receptor LRP-1/RAGE related to transport Aβ,so as to clarify the effect of bushenhuoxue method on the expression of LRP-1/RAGE protein in brain microvascular endothelial cells induced by glucolipotoxicity.Results:1.CCK-8 was used to detect cell proliferation,and the low,medium and high doses of bushen prescription were finally selected as 25mg·L^-1,50mg·L^-1,100mg·L^-1,huoxue prescription were 5mg·L^-1,10mg·L^-1,20mg·L^-1,bushenhuoxue prescription were 30mg·L^-1,60mg·L^-1,120mg·L^-1.When BEND3 cells were stimulated by HG and PA,the cell viability increased from（100±2.3）%to（62.6±0.47）%（P<0.001）.After bushen prescription(BS,25mg·L^-1,50mg·L^-1,100mg·L^-1),huoxue prescription(HX,5mg·L^-1,10mg·L^-1,20mg·L^-1)and bushenhuoxue prescription(BSHX,30mg·L^-1,60mg·L^-1,120mg·L^-1)intervention after the cell vitality,respectively up to（69.4±2.2）%（P<0.05）,（70.2±1.2）%（P<0.05）,（71.3±1.3）%（P<0.01）,（69.7±1.4）%（P<0.05）,（70.0±2.8）%（P<0.05）,（72.2±2.8）%（P<0.01）,（73.1±5.3）%（P<0.01）,（75.8±3.1）%（P<0.01）,（77.8±4.4）%（P<0.001）;2.BEND3 cells stimulated by HG and PA significantly increased the content of relevant indicators of oxidative stress and inflammation.After the intervention of bushen formula(BS,25mg·L^-1,50mg·L^-1,100mg·L^-1),huoxue formula(HX,5mg·L^-1,10mg·L^-1,20mg·L^-1)and bushen huoxue formula(BSHX,30mg·L^-1,60mg·L^-1,120mg·L^-1),the contents of oxidative stress factors（ROS/VEGF/NO/iNOS/MMP-2/MMP-9/TIMP-1）and inflammatory factors（IL-6/ICAM-1/TNF-α/TGF-β1）decreased were decreased（P<0.05 or P<0.01 or P<0.001）;3.BEND3 cells suffered from endoplasmic reticulum stress,GRP78,p-eIF2α,ATF4 and NF-κB expression significantly raised,give bushen formula(BS,100mg·L^-1),huoxue formula(HX,20mg·L^-1)and bushenhuoxue formula(BSHX,120mg·L^-1)after the intervention GRP78,p-eIF2α,ATF4 and NF-κB protein were significantly decreased（P<0.05 or P<0.01）.4.High glucose and hyperlipemia after stimulation,BEND3 membrane transport Aβrelated LRP-1/RAGE receptor protein expression changes,which is responsible for Aβoutward transport receptors in the brain LRP-1 protein expression significantly lower,and transports Aβinto the brain the RAGE receptor protein expression was significantly increased,giving bushenhuoxue formula the LRP-1 protein expression increased after the intervention,the RAGE protein expression decreased（P<0.05 or P<0.01）.Conclusion:1.Bushen formula(BS,25mg·L^-1,50mg·L^-1,100mg·L^-1),huoxue formula(HX,5mg·L^-1,10mg·L^-1,20mg·L^-1)and bushen huoxue formula(BSHX,30mg·L^-1,60mg·L^-1,120mg·L^-1)had a protective effect on the cell damage model of diabetic cerebrovascular lesions stimulated by HG and PA;2.Bushenhuoxue method could improve oxidative stress,inflammatory reaction and endoplasmic reticulum stress caused by diabetic cerebrovascular lesions;3.Bushenhuoxu method could regulate the expression of LRP-1/RAGE protein,which is related to the transport of Aβ.