The Neuroprotective Effects Of Hsp20 Against Oxygen-glucose Deprivation Followed By Reperfusion And Its Underlying Mechanism

Chapter I:Expression Pattern of Hsp20 in Mouse N2A Cells upon OGD/R treatmentObjective:To elucidate alteration of cellular viability and apoptosis, morphology of mitochondria and Golgi apparatus in mouse N2A cells upon oxygen-glucose deprivation followed by reperfusion (OGD/R),as well as expression pattern of Hsp20.Methods:We employed OGD/R model to mimic ischemic-like conditions in vitro. Cellular viability and apoptosis were measured using the MTT assay and flow cytometry. The morphologic change of mitochondria and Golgi apparatus was determined by immunofluorescence.GM130 and Hsp20 expression levels were determined by Western blot and quantitative Real-Time PCR.Results:1. Treatment with OGD/R markedly reduced cellular viability in mouse N2A cells at 12-hour and 24-hour time points recovery from 4-hour OGD (P<0.01), while increased apoptosis rate at 6-hour,12-hour and 24-hour time points recovery from 4-hour OGD (P<0.05)2. Expressions of Hsp20 were strongly downregulated in mouse N2A cells at 0-hour and 6-hour time points recovery from 4-hour OGD (P<0.05), both at mRNA and protein levels, and they returned to basal level 12 and 24 hours after OGD treatment.3. The protein levels of total and phosphorylated Hsp20 decreased to approximately the same extent at the 0-hour and 6-hour recovery time points following 4 hours of OGD. However, the ratio of phosphorylated to total Hsp20 protein was significantly higher than control 12 and 24 hours after OGD treatment (P<0.05)4. Upon OGD/R, compared to control, expressions of GM130 and the morphology of Golgi apparatus showed no significant differences. However, OGD/R induced mitochondria fragmentation. Many filamentous mitochondria converted small and round organelles following OGD/R.Conclusion:Cellular viability and apoptosis, as well as morphology of mitochondria damaged greatly in mouse N2A cells upon OGD/R treatment. Meanwhile, the expression pattern of Hsp20 was regulated upon OGD/R treatment. However, expressions of GM130 and the morphology of Golgi apparatus were not affected. Chapter?Construction and Expression of Hsp20 and its mutantsObjective:To construct expression plasmids of Hsp20 and its constitutively dephosphorylated mutant Hsp20s16A, as well as the constitutively phosphorylated mutant Hsp20s16DMethods:Total RNA was isolated from N2a cell cultures and reverse transcription was then performed. Hsp20 was obtained by using primers with Restriction enzyme EcoR I and BamH I in 5'-Terminal. The PCR products were cloned to pEGFPNl vector using EcoR I and BamH I. After being identified by restriction enzyme digestion and sequencing, the recombinant plasmids were transfected into N2a cells. Hsp20 expression in the transfected cells was assayed by immunofluorescence and immunoblotting.Results:We obtained CDS sequences of Hsp20 successfully by PCR. Enzyme digestion and DNA sequencing showed that the CDS sequences of Hsp20 were correctly inserted into pEGFPN1 vector. Same expression and distribution of Hsp20 and its mutants in N2a cells were confirmed by immunofluorescence. Expressions of Hsp20 and its mutants were confirmed by immunoblotting and their molecular weights were correct.Conclusion:We successfully constructed expression plasmids of Hsp20 and its constitutively dephosphorylated mutant Hsp20s16A, as well as the constitutively phosphorylated mutant Hsp20S16D. Chapter?The Neuroprotective Effects of Hsp20 and its Underlying MechanismObjective:To elucidate the neuroprotective effects of Hsp20 against oxygen-glucose deprivation followed by reperfusion (OGD/R) and its potential mechanism.Methods:Mouse N2A cells were transfected with pEGFP-Hsp20, or pEGFP-Hsp20 (S16D) or pEGFP-Hsp20(S16A) for 36 h, and then treated with 12-hour reperfusion following 4-hour OGD. Cellular viability and apoptosis were measured using the MTT assay and flow cytometry. The morphologic change of mitochondria was determined by immunofluorescence. Bax, Bcl-2 and cytochrome c expression levels were determined by Western blot.Results:1, Compared with the control, the cellular viability was significantly higher in N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D (p<0.05). N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D also displayed a significant decrease in the number of apoptotic cells upon OGD/R exposure (p<0.01).2. pEGFP-Hsp20 (S16A)-transfected cells exhibited no significant alteration in OGD/R-induced apoptosis and cellular viability, compared with the control.3. N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D displayed much less damage to the structure of mitochondria. However, pEGFP-Hsp20 (S16A)-transfected cells exhibited no significant difference compared with the control.4. N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D displayed a marked decrease in the expression of Bax and an increase expression of Bcl-2. N2A cells transfected with pEGFP-Hsp20 or Hsp20s16D also inhibited cytochrome c released from mitochondria into cytosol upon OGD/R (p<0.05). However, pEGFP-Hsp20 (S16A)-transfected cells exhibited no significant alteration in OGD/R-induced expression of Bax and Bcl-2 and failed to inhibit OGD-induced cytochrome c release. Conclusions:1. Hsp20 displayed neuroprotective effects against oxygen-glucose deprivation followed by reperfusion.2. Phosphorylation of Ser16 played an important role in the neuroprotective effect of Hsp20. Otherwise, blockade of Hsp20 phosphorylation abrogated its neuroprotective effect.3. Neuroprotective effects of Hsp20 were mediated by recovering mitochondria from the OGD/R damage.4. Neuroprotective effects of Hsp20 on OGD/R were also mediated through inhibition of mitochondrial apoptotic signaling pathways.