Antagonism And Mechanism Of Tan On Upregrulation Of Activity Of NF-κ B Induced By LPS

Objective:Lipopolysaccharide(LPS) is the main ingredient of the plasmalemma of the gram-negative bacteria and the main cause of the Inflammatory reaction of animals or humain beings. Toll like receptor4(TLR4) is the main receptor of LPS. LPS can induce NF- k B activation through TLR4 and its activation can cause a series of expression of Inflammatory reaction correlated genes, release some cytokines and Inflammatory mediators, activate the acquired immune system, cause the local or systemic inflammatory responsesyndrome.Atherolerotic(ATH)is the most popular and crucial one of arteriosclerotic vascular diseases. When ATH was formation, it occur from the tunica intima to the adventitia, including many symptoms such as proliferation of smooth muscle, formation of connective tissue Matrix, assemblage of lipid et al. Immunue and inflammatory responses have play a key role in the occurrenc and development of ATH just as Ross has pointed out that ATH is an inflammation. Kiechl and his group also said that a people with Asp299Gly Gene type of TLR4 is much more resistant to inflammatory reponses and has a lower morbidit of ATH. It not only provides a novel theory to the pathogenesis of ATH, but also develops a new clinically relevant therapeutic target for ATH.Dingxi recipe (DXR) is formed with the theory of traditional Chinese medicine (TCM). It shows great therapeutic effects in preventing and treating ATH and IHD in clinical Application. Our previous studies show: DXR can deduce the expression of CD41 and CD62p, repress the platelet aggregation. However, the precise mechamism(s) remains to be elucidated. [Salvia Miltiorrhiza Bunge] is one of the major compositions of DXR. Current pharmacological study demonstrates that Tanshinone, Tan, the major active substance of [ Salvia Miltiorrhiza Bunge ] , is consisted of ten momoners; Clinical and experimental evidence indicatesTan mediates vasodilation of coronary, and inhibits platelet aggregation and thrombus formation. It also attenuates growth and proliferation of vascular smooth muscle cells. In general, Tan shows great therapeutic effects in preventing and treating in8atherosclerosis and IHD. Now it is widely used in clinic with many types of preparation. Both interior and exterior NO mediate vasodilation of all kinds of vessels, including artery, vein, coronary et al. Tan can enhance the release of NO from the cultured porcine aorta endothelial cells. In summary, Tan can decrease the activation of NF-KB induced by LPS. Recently, a lot of hypothesis about Tan inhibiting the inflammatory response induced by LPS on cell level has been revealed. To observe the effects of LPS induced NF-KB activity of the HEK293 cell and CHO-kl cell and the antagonistic action of tanshinone (Tan) This work will primarily demonstrate the compounded Chinese herbs DXR how to effect the LPS induced NF-KB activity on signaling transduction.level, so provide experimental and clinical evidence for it in preventing and treating ATH and IHD.Methods: With the assistant of lipofectamine-mediated co-transfection technique, pNF-KB-TA-Luc, pCMV-p were cotransfected into HEK-293cells The HEK-293 cell was treat with LPS or Tan. NF-KB activity was measured by pNF-icB-TA-Luc report gene. After infections of the CHO-K1 cells with the resultant viruses carrying TLR4 and ATLR4 gene pNF-xB-TA-Luc, pCMV-p were cotransfected into CHO-K1 cells with the assistant of lipofectamine-mediated co-transfection technique. NF-KB activity induced by LPS and Tan was measured by pNF-KB-TA-Luc report gene.Results :1. Double restriction enzyme digestion and sequencing both confirmed that the recombinant plasmids, pAd-TLR4 and pAd-ATLR4, were constructed correctly. Rrecombinant adenovirus Ad-TLR4 and Ad-ATLR can express effectiviy in CHO-K1 cell and HEK-293 cell.2. It can be seen from the results of the report gene that LPS can enhance the activation of NF-KB in HEK-293cells and Tan can decrease the activation of NF-KB induced by LPS in HEK-293 cells. TLR4 recombinant adenovirus activated the activation of N...