Screening Of TNF-α Inhibitors And C₂D Mechanism Study

TNF-a (Tumor necrosis factor-a) is a kind of cytokine with a broad range of biological activities. The excessiveness of TNF-a in tissues and leads to many inflammatory diseases. Now most of the anti-TNF drugs in clinic have carcinogenic side-effect or have other disadvantages, so finding efficient and TNF-a inhibitors to reduce the production of TNF-a or inhibit the activity of downstream protein would have therapeutic effect on inflammatory diseases.Firstly, this thesis uses the L929cell line to establish a TNF-a inhibitor screening model. In total34compounds in1031compounds tested with this model displayed the effect of improving the survival rate of L929to more than50%at the concentration of10μM and20μM, when TNF-a’s concentration is3ng/mL. Among the34compounds,5natural products and l synthetic compound showed best effect, they would hav potency of becoming the promising lead compounds.Secondly, in this paper we choosed a lead compound C2D and studied its mechanism of anti-TNF with CCK-8assay、FCM、Western blot, et, al. We got the results as follows:(1) C2D has no cytotoxic effect on L929cell line even concentration of100μM. On the contrary, it can protect cells when TNF-a is added in culture medium.(2) C2D protected L929cell line by reducing ROS through affecting the production of two upstream proteins: p38. It increased production of the p-p38.Thirdly, we found that in the presence of TNF-a, the amount of RIP3in L929cell line decreased at90min, then increased quickly. Through Western blot、 Real-time qPCR and co-IP methods,we got the following conclusions:(1) During the previous1h-2h, there has no obvious variation on the RNA expression level, but the amount of ubiquitined RIP3increased compared with control.(2) During this period, the interation between RIP land RIP3was detected todecrease.(3) It’s possible that RIP3is cleaved by caspase-8at that point.(4) During this period, more NF-κB entered into nucleus.We can suggest from above that under the action of TNF-α, L929cell lines iniciatated the self-healing mechanism to counteract the necrotic effect of TNF-α by decreasing the bingding of RIP1and RIP3, for it is the switch from apoptotic cell death. With the action of TNF-α continued, the balance switched to necrosis.In this paper, we screened TNF-α inhibitors and researched TNF-α downstream mechanism.These results will make positive contribution to develop independent intellectual property rights of TNF-α inhibitor drugs.