Chloride Channel Blockers' Down-regulating Effect On Aβ Induced Apoptosis And Its Relationship With JNK Signal Transduction Pathway

AD is a common degenerative disease of the central nervous system .Its causes and mechanism are still unclear. A number of experiments indicate that Aβinduced apoptosis is one of the pathophysiology mechanisms by which Aβinduces AD.Chloride channel is a kind of important channel in the cellular membrane by which apoptosis can be mediated. We will investigate what role the chloride channel plays when PC12 apoptosis is induced by Aβ25-35.MAPK signal transduction pathway can perform significant functions in cell apoptosis. JNK signal transduction pathway is an important branch of MAPK signal transduction pathway. We will try to find out what role JNK plays when PC12 cells apoptosis is induced by Aβ25-35 ,also ,we will try to find out the relationship between chloride channel blockers and JNK.The mechanisms by which Aβinduces cell apoptosis are very complex, People are still unclear about the key point that can be made use of to prevent cell apoptosis. So if the mechanism and the key point are known, it is possible that AD can be cured.Aims:1. To investigate whether DIDS can inhibite PC12 apoptosis induced by Aβ25-35.2. To investigate whether Phloretin , a relatively specific VSOR Cl- channel blocker , can inhibite PC12 apoptosis induced by Aβ25-35.3. To find out what role JNK plays when PC12 apoptosis is induced by Aβ25-35 and to find out the relationship between chloride channel blockers and JNK.Methods:1. Serial subcultivation PC12 cells were the objects for research , cells were divided into different groups: the negative control group(normal),positive group(treated with Aβonly) and experimental groups(treated with Aβ+chloride channel blockers DIDS or Phloretin).2. Cell survival rate was estimated by MTT , Fluorescence microscope was used to observe the morphological change of the nuclear after stained by Hoechst33258 , the integrity of cell member was surveyed by measuring released LDH level, apoptosis was further proved by agarose gel electrophoresis of DNA.3. The expressions of P-JNK of PC12 cells induced by Aβon different time points were detected by Western Blot.The expressions of P-JNK in different groups were detected by Western Blot.4. All data were analyzed by SPSS11.0.Results: 1. The experiment results indicated that apoptosis occurred when PC12 cells were treated by Aβ. The experiment results also indicated that DIDS and Phloretin could protect cells from apoptosis induced by Aβ.2. MTT manifested low cell survival rate(58.4%±9.3%) when cells were exposed to 40μmol/L Aβ25-35, cell survival rates in experimental groups were : Aβ25-35+DIDS(86.0%±7.2%) and Aβ25-35+ Phloretin (94.1±9.3%), and the difference possessed statistical significance(P<0.01).3. Hoechst33258 displayed obvious cell shrinkage , and the breakage , aggregation , pyknosis of nuclear chromatin when cells were exposed to 40μmol/L Aβ25-35 and the number of cells with typical apoptosis morphological changes decreased in the experimental groups.4. The level of released LDH : the difference between the negative group(328.7±18.9 U/L) and positive group(433.1±41.5 U/L)possessed statistical significance (P < 0.01);the differences between the positive group and experimental groups (Aβ25-35+Phloretin:354.4±34.3 U/L and Aβ25-35 + DIDS:366.7±28.3 U/L) both possessed statistical significance(P < 0.01).5. Agarose gel electrophoresis of DNA manifested obvious DNA Ladder in the positive group , while none could be seen in the negative group or experimental groups .6. P-JNK expression after PC12 cells had been exposed to Aβ25-35 for 6h was increased compared with that of 0h and the difference possessed statistical significance(P < 0.01).Also ,the differences of P-JNK expressions between two experimental groups and the positive group meant statistical significance(P < 0.01). Conclusion:Both DIDS and Phloretin could prohibit Aβ25-35 induced PC12 apoptosis and the protective role may correlate with down regulating the phosphorylation of JNK. As Phloretin , a relatively specific VSOR Cl- channel blocker , could protect cells from apoptosis , it was supposed that VSOR Cl- channel was activated when PC12 cells were exposed to Aβ.Also, it was supposed that chloride channel blockers exerted protective role by inhibiting the phosphorylation of JNK.