Neuroprotective Effects Of The PDE4 Inhibitor FCPR16 Against TNF-α-induced Neuronal Injury

Objective:Neuroinflammation is one of the most common pathological characteristics during the process of acute neurological disorders and neurodegenerative diseases,such as stroke and Alzheimer’s disease.Under the pathological conditions that nerve cells are infected by microorganisms or stimulated by various endogenous stimuli,microglial cells are activated,and thereby promoting the production of pro-inflammatory cytokines.Phosphodiesterase 4(PDE4)is highly expressed in microglial cells.Inhibition of PDE4 produces anti-inflammatory effects through decreasing the transcription and cleavage of inflammatory cytokines.However,whether PDE4 inhibitor could directly protect against neuronal damage induced by inflammatory factors has not yet been well studied.FCPR16,a novel PDE4 inhibitor synthesized in our laboratory,shows a higher selectivity for PDE4D.Our previous studies indicated that FCPR16 reduced the proportion of M1 type microglia in the hippocampus of mice subjected to chronic unpredictable mild stress,and thereby reducing the production of inflammatory factors.While whether FCPR16 is able to attenuate neuronal damage induced by inflammatory factors is unclear.Lipopolysaccharide(LPS),a bacterial cell wall component,is the canonical agent used for the construction of inflammatory model.In view of the fact that the inflammation of neurological diseases is mostly aseptic inflammation,which is characterized by a large increase in inflammatory factors.Therefore,we intend to directly use tumor necrosis factor-α(TNF-α)to induce neuronal damage.In this study,we aimed to investigate the protective effects of FCPR16 against TNF-α-induced neuronal damage in HT-22 hippocampal neuronal cells and explore the possibly involved mechanisms.Methods:(1)HT-22 neuronal cells were exposed to various concentrations of TNF-α(0.1-100 ng·mL-1)for 24 h,and Cell Counting Kit-8(CCK-8)assay was used to investigate the toxic effects of TNF-α in neuronal cells.We then pretreated cells with FCPR16(1.5-100 μM)for 24 h in the present of TNF-α(1.5 ng·mL-1).Cell viability was determined by CCK-8 assay.(2)The apoptotic rate of HT-22 cells was determined by flow cytometry,nuclear morphology was observed by Hoechst staining,and the ratio of cell death was detected by PI/calcein double staining.Western blot was used to investigate the levels of apoptosis-related proteins.The above parameters were used to evaluate the anti-apoptotic effects of FCPR16 in HT-22 neuronal cells.(3)Silencing PDE4B was used to further confirm the anti-apoptotic effects of PDE4 inhibition in HT-22 neuronal cells in response to TNF-α.HT-22 cells were transfected with PDE4B siRNA using Lippofectamine 2000.24 h latter,cells were exposed to TNF-α(1.5 ng·mL-1)for 12 h.The protective effects of siPDE4B was then detected by Western blot and PI/calcein double staining,respectively.(4)Evaluation of the effect of FCPR16 on the expression of synapse-related proteins in HT-22 cells.Cells were pretreated with FCPR16 for 1 h,and then were incubated with TNF-α(1.5 ng·mL-1)for 9 h.The levels of GAP-43,synapsin 1 and BDNF were detected by Western blot(5)Investigation on the possibly involved signaling pathways.① HT-22 cells pre-treated various concentrations of FCPR16(12.5-50 μM)for 1 h were incubated with TNF-α for 30 min.The levels of the p-JNK and t-JNK were detected by Western blot.② Cells were pre-treated with FCPR16 or JNK inhibitor SP600125(1 μM)for 1 h,and then were incubated with TNF-α for 12 h.Cytotoxicity and the levels of apoptosis related proteins was detected by the lactate dehydrogenase(LDH)assay and Western blot,respectively.③HT-22 cells pre-treated with 50 μM FCPR16 were incubated with TNF-α for 1 h.The translocation of NF-κB p65 was detected by immunofluorescence straining and Western blot.④After pre-treatment with FCPR16(50 μM)for 1 h,HT-22 cells were incubated with TNF-α(1.5 ng·mL-1).The expression level of iNOS and ROS was detected respectively by Western blot and CellROX Deep Red Reagent.The levels of MDA and 3-NT were detected respectively by a lipid peroxidation MDA assay Kit and 3-NT Elisa Kit.⑤ Western blot and immunostaining were used to detect the levels of endoplasmic reticulum stress related proteins.Results:(1)TNF-α caused cell death in HT-22 neuronal cells in a concentration-dependent manner,with approximately 50-60%of the cells left viable at 1.5 ng·mL-1.FCPR16,in a concentration-dependent manner,protected HT-22 cells against cell death induced by TNF-α.(2)Inhibition of PDE4 by FCPR16 significantly decreased the rate of apoptosis,ameliorated nuclear morphology and attenuated the levels of Cleaved caspase 3 and Cleaved caspase 8 induced by TNF-α in HT-22 neuronal cells.(3)Silencing PDE4B decreases cell death rate and the expression of apoptosis-related proteins in HT-22 neuronal cells in response to TNF-α.(4)HT-22 cells were treated with 1.5 ng·mL-1 TNF-α for various time,TNF-αdecreased the expression of synapse-associated proteins in a time-dependent manner.After treatment with FCPR16,the expression levels of GAP-43,Synapsin 1 and BDNF increased significantly。(5)Compared with TNF-α-treated group,FCPR16 significantly reduced the phosphorylation of JNK in a dose-dependent manner.JNK specific inhibitor SP600125 significantly decreased LDH activity and the expression of apoptosis-related proteins.FCPR16 decreased the translocation of nuclear factor kappa-B(NF-κB p65)from the cytosol into the nucleus.In addition,FCPR16 decreased the expression of iNOS,the production of ROS and the levels of 3-NT and MDA in HT-22 cells exposed to TNF-α.(6)TNF-α had no effect on the expression of glucose regulated protein78 kD(GRP78)and the phosphorylation of eukaryotic initiation factor 2α(eIF2α).Immunostaining showed that both FCPR16 and TNF-α did not affect the fluoresence intensity of p-eIF2α.Conclusion:TNF-α reduced cell viability and promoted apoptosis in HT-22 cells,while inhibition of PDE4 by FCPR16 blocked the role of TNF-α,enhanced cell viability and reduced synaptic damage.Suppression of JNK and NF-κB/iNOS signaling is probably involved in the protection of FCPR16.