Effect Of 15-LOX/15-HETE On The Expression Of Kv Channels In Cerebral Vasoconstriction Induced By Hypoxia

Objective The water fall effect of AA participate the patho-course of reperfusion injury after cerebral ischemia.The metabolism of AA by 15-LOX can generate 15-HETE,and 15-LOX has expressed in brain tissue,the latter can produce 15-HETE.Our previous research has proved that the expression of 15-LOX in CASMCs increased when under the hypoxia condition;the production of 15-LOX is 15-HETE,which can contract carotid artery by inhibiting the expression and function of Kv channels.Our experiment will Knockout and Over-express 15-LOX gene of CASMCs by RNA interference,and add NDGA the inhibitor of 15-LOX.Observe the effect on Kv channels? expression and function,and the production of 15-HETE in CASMCs.To prove that 15-LOX can down regulate Kv channels by its production 15-HETE in hypoxia brain injury.Observe whether 15-LOX?s inhibitor can affect the Kv channels? expression and function in hypoxia CASMCs,and prove that 15-LOX?s inhibitor can relieve the vasoconstriction in ischemic cerebrovascular disease.Ultimately provide 15-LOX a new target for the treatment of ischemic cerebrovascular disease.Methods 1?Twenty healthy Wistar rats were executed,and cerebral arteries were isolated to make into vascular ring.Two rings were made from every rat,and randomly divided into five groups in average(n=8): group A(control group): the vascular ring wre placed in normoxic water bath for 2h with normal oxygen supply;group B(hypoxia group): with low flow oxygen supply;group C(hypoxia group with 10?mol/L NDGA): added 10?mol/L NDGA and with low flow oxygen supply;group D(hypoxia group with 20?mol/L NDGA): added 20?mol/L NDGA and with low flow oxygen supply;group E(hypoxia group with 30?mol/L NDGA): added 30?mol/L NDGA and with low flow oxygen supply.Added 15-HETE to each group?s KH liquid,the density of 15-HETE from 10-8 mol/L to 10-6mol/L.Observed the change of vascular ring?s construction level,and which density of NDGA can inhibit 15-LOX best.2?Healthy Wistar rats were decapitated and CASMCs were cultured by enzyme digestion.All CASMCs were divided into five groups: group A(control group): CASMCs were cultured in normal condition for 48 h with normal oxygen supply;group B(hypoxia group): with low flow oxygen supply;group C(over-expressing 15-LOX group): CASMCs 15-LOX gene were Over-expressed;group D(hypoxia group with knockout 15-LOX): CASMCs 15-LOX gene were knockout and with low flow oxygen supply;group E(hypoxia group with NDGA): added NDGA and with low flow oxygen supply.ELISA was used to detect the production of CASMCs 15-HETE;RT-PCR and western-blot techniques were used to detect the expression of m RNA and protein of CASMCs Kv1.5?Kv2.1;Voltage-clamp was used to detect the channel currents of CASMCs Kv1.5?Kv2.1.Results 1?The tension of ICA rings increased significantly when under the hypoxia condition,and the increasing degree of tension was significantly inhibited when added NDGA,between the control group and hypoxia group.The optimal concentration of NDGA was 10?mol/L;2?The production of 15-HETE and the expression of Kv1.5?Kv2.1 were Significantly different from control group,so did the Kv currents.? The production of 15-HETE increased and the expression of m RNA and protein of Kv1.5?Kv2.1 down regulated in hypoxia group and Over-expressing 15-LOX group,compared with control group,the Kv currents also were inhibited(P?0.01);? The production of 15-HETE decreased and the expression of m RNA and protein of Kv1.5?Kv2.1 up regulated in hypoxia group with knockout 15-LOX and hypoxia group with NDGA,compared with hypoxia group,the inhibition degree of Kv currents depressed(P?0.01).Conclusions The important mechanism of hypoxia brain injury was the inhibition from 15-LOX and its production 15-HETE to expression and function of Kv channels;15-LOX?s inhibitor NDGA can relieve the cerebral vasoconstriction induced by hypoxia;We can consider 15-LOX and its inhibitor NDGA as target to study the new method of ischemic cerebral vascular diseases? treatment.