Research Of Molecular Target Of Ox-LDL Induced Endothelial Cell Injury Based On Protein Microarray Technique And Discovery Of Potential New Drugs Of Chinese Traditional Medicine

Objective:1. To investigate the potential signaling pathway and molecular target in Atherosclerosis development caused by endothelial cell injury by analyzing two key events, apoptosis related protein and cytokine level, in Ox-LDL induced endothelial injury with protein micro-array technique. And to confirm the result and in vivo by an ApoE-/-mice mode for further study of the molecular mechanism of drugs against atherosclerosis.2. To study the anti-Atherosclerosis mechanism of major activity ingredients in "Fu Fang Guan Xin Dan Shen Fang" proved in clinical research at molecular level. To study the synergy effect of these compounds by median effect equation in an endothelial cell injury mode. And to re-develop proprietary Chinese traditional medicines and promote the modernization of Chinese traditional, medicine by investigating the mechanism with networkpharmacology.3. Screen the activity of10Flavonoids ingredients and study anti-Atherosclerosis mechanism of a high activity component, Myricitrin, at molecular level. To provide a scientific basis of discovery and development of new drugs in Chinese traditional medicineMethods1. Multiple methods(cell viability, FACS, TUNEL,ΔΨm) was adopted to evaluate the injury of HUVES induced by ox-LDL. An apoptosis associated protein micro-array (43kinds) and an inflammatory factor micro-array (40kinds) were adopted to investigate the target of protein involving in this injury process. And the results are confirmed in an ApoE-/-mice mode.2. Screen the ROS savage activity of10Flavonoids and19active ingredients in ’The Guan Xin Dan Shen Fang" by an endothelial cell injury mode. And the synergistic action of protection in endothelial cells injury of Salvianolic acid B and Notoginseng triterpenes R1was evaluated under the guide of network pharmacology and median effect equation.3. Pathology Technology(Oil red O staining. Immunohistochemistry), serum biochemical(Blood lipids, inflammatory factors, lipid peroxidation products, antioxidant enzymes), molecular cell biology technology(western blot, RT-PCR, EMSA) and in vivo imaging techniques(ultrasonic, mico-CT, LPO inflammatory probe fluorescence imaging) were adopted to assess the protection capacity of Myricitrin(Flavonoids compound) and Notoginseng triterpenes Fl(from "Guan Xin Dan Shen Fang") to endothelial cell injury at cell level and animal level of an AS mode in ApoE-/-mouse.Results:1.1.1The injury of HUVECs induced by Ox-LDL is in a dose and time dependent manner. And the optimal concentration of ox-LDS is70μg/ml and the duration of treatment is24hrs. The cell viability decreased to50%,30%cells suffered apoptosis, ΔΨm is declined.1.2The protein micro-array of apoptosis associated proteins indicated that, in HUVECs, the expression level of proteins of Bcl-2family (Bax, Bad and Bim), IAP family (XIAP, cIAP1/2and surivivin), HSP family (HSP27/60/70) and IGF family (IGFBP-1/2) changes after ox-LDL treatment.1.3The protein micro-array of inflammatory factors showed that, in the supernatant of cultured HUVECs, G-CSF, M-CSF, MIP-1δ, ICAM-1, IFNy, MIG, TNF-a and its receptor TNFR1, IL-1, IL-4, IL-6, IL-11, IL-12p70, IL-12p90, IL-15and IL-16was up-regulated after the exposure to ox-LDL.2.2.1Activity screening by the established platform revealed that:Salvianolic acid A/B, Rosmarinic acid, Tanshinone IIA Sulfonate, Notoginseng triterpenes R1/R2, ginsenoside Rgl/F1, Gypenoside XVII, Luteolin, Luteoloside, had a protection activity of endothelial injury.2.2The combination of Salvianolic acid B and Notoginseng triterpenes R1which is abundant in "Guan Xin Dan Shen Fang" had a synergetic action; the protection activity is much higher than single drug treatment. And the ratio of Salvianolic acid B and Notoginseng triterpenes R1at3:1achieved the highest activity.2.3Administration of Notoginseng triterpenes F1(50mg/kg, i.g.) to a high fat diet induced Atherosclerosis mode of ApoE-/-mouse for6weeks reduced the area of AS plaque. And in vivo LPO inflammatory probe imaging showed that Fl strongly inhibited inflammation response in a hyperlipidemia condition. In vitro resulst indicated that Notoginseng triterpenes F1was able to inhibit the attachment of HUVECs induced by human monocyte THP-1, and reduce the expression of LOX-1and TLR-4mediated by ox-LDL. It also blocked the nuclear localization of NF-κ B, down-regulated the expression of TNF-a, IL-6, G-CSF, MIP-1δ, ICAM-1, MIG, IL-1, IL-15and IL-16.3.3.1Activity screening by the established platform revealed that:Myricetin, Myricitrin, Dihydromyricetin and Isorhamnetin had a protection activity of endothelial injury.3.1In the Atherosclerosis mode of ApoE-/-mouse receiving high fat diet, the treatment of Myricitrin(50mg/kg, i.g.) for6weeks inhibited the formation of AS plaque. The result was confirmed by ultrasonic imaging, micro-CT of calcification, oil Red O staining, TUNEL staining of endothelial cells and Caspase3activity assay. Although serum level of HDL-C, LDL-C, TC was not changed after Myricitin treatment, the serum level of ox-LDL, inflammatory factor(MCP-1, sICAM-1and CRP), lip id peroxidation products (MDA and4-HNE) and antioxidant enzyme (CAT, SOD, GSH-px) was down-regulated by the administration of Myricitrin. In vitro results indicated that Myricitin was able to reduce apoptosis of HUVECs induced by ox-LDL in a dose dependent manner. The mechanism of this protection was associated with the down-regulation of the expression of LOX-1and ROS release by its activation. Besides, the treatment of Myricitrin also activated PI3K7Akt/eNOS and Stat3/HO-1signaling pathway, which down-regulated pro-apoptosis factors of Bax and up-regulate anti-apoptosis factors of XIAP, cIAP-2and surviving, preventing cell death.Conclusion:1. The result of protein micro-array indicated that the effect of ox-LDL caused endothelial cell injury by the combination of oxidative response, inflammation induced cell apoptosis, functional disorder. And anti-oxidative, mitochondrial associated apoptosis, cell proliferation and inflammation signaling pathways are involved in this EC damage.2. The activity screening of Flavonoids and active ingredients in "The Guan Xin Dan Shen Fang" by the established platform revealed that the protection activity is associated with their anti-oxidative effect and/or anti-inflammation activity. This result suggested the potential value of anti-Atherosclerosis in natural product in massive natural material and the bright future of the therapeutically potential against comprehensive diseases.3. The combination of Salvianolic acid B and Notoginseng triterpenes R1which is abundant in "Guan Xin Dan Shen Fang" had a synergetic action; the protection activity is much higher than single drug treatment. And the ratio of Salvianolic acid B and Notoginseng triterpenes R1at3:1achieved the highest activity.4. Myricitrin was able to attenuate the development of AS by reducing the apoptosis of endothelial cell. It was capable to inhibit LOX-1expression and ROS releasing mediated by LOX-1. And pro-apoptosis protein like Bax and bad is down-regulated, anti-apoptosis protein XIAP, cIAP-2and Survivin through PI3K/Akt/eNOS and Stat3/HO-1signaling pathway by Myricitrin treatment.5. Administration of Notoginseng triterpenes F1protected endothelial cell injury and anti-AS by reducing inflammation response of ES. The mechanism of this effect is by inhibiting the attachment of HUVECs induced by human monocyte and reduce the expression of LOX-1and TLR-4mediated by ox-LDL. It also blocked the nuclear localization of NF-κ B, down-regulated the expression of multiple inflammation factors.