Experimental Study On Inhibitory Effect Of Insulin-Like Growth Factor-1 Against Inflammatory Response In Microglia

Objective:Insulin-like growth factor-1（IGF-1）,a neurotrophic factor in the brain,can be produced by the liver and enter the brain through the blood-brain barrier.In the central nervous system,IGF-1 can be produced by neurons and glial cells.It has been found that there is an interaction between insulin-like growth factor-1 receptor（IGF-1R）and estrogen receptor signal pathway.In breast cancer,IGF-1 induces the migration and proliferation of cancer cells by regulating the expression and function of the G protein-coupled estrogen receptor（GPER）and triggering the activation of intracellular signal molecules.Our previous studies have shown that IGF-1can inhibit 1-methyl-4-phenylpyridinium（MPP⁺）-induced inflammatory response of rat mesencephalic astrocytes and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）/MPP⁺-induced damage of dopaminergic neurons.The mechanism is related to GPER-mediated signaling.However,it is not clear whether GPER is involved in the inhibitory effect of IGF-1on microglial inflammation.Using BV2 microglial cells and primary microglia,the present study aimed to investigate the effect of IGF-1 on phenotypic polarization of microglia and identify the possible signal pathway of IGF-1 against the microglial inflammatory response by using pharmacological blockade,si RNA interference,and GPER gene knockout mice.Our results will provide a theoretical basis and experimental data for the anti-inflammation as well as neuroprotective effect of IGF-1.Methods:1.The inflammatory cell models of BV2 microglia and primary microglia from GPER^+/+（wild type）and GPER^-/-（gene knockout）C57BL/6J mice were induced by lipopolysaccharide（LPS）;2.The cell viability of BV2 microglia was detected by MTT assay;3.Real-time PCR（RT-PCR）was used to detect the m RNA expressions of Arg-1, interleukin-10（IL-10）,interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,inducible nitric oxide synthase（i NOS）and cyclooxygenase（COX-2）;4.The protein expressions of i NOS,COX-2,p-ERK/ERK,p-Akt/Akt and GPER were detected by Western blot;5.The anti-inflammatory effect of IGF-1 was observed by using pharmacological blockade and si RNA interference to target the signal pathway of IGF-1R and GPER.Results:1.MTT results showed that LPS（1μg/m L）and different concentrations of IGF-1（6.25, 12.5,25,50 ng/m L）had no significant effect on the survival and proliferation of BV2 microglia（P>0.05）.2.LPS could remarkably enhance the m RNA levels of M1 pro-inflammatory cytokines TNF-α,IL-1β,COX-2 and i NOS in BV2 microglia（P<0.01）.Pretreatment with different concentrations of IGF-1（12.5,25,50 ng/m L）could inhibit the gene expressions of the above inflammatory cytokines induced by LPS（P<0.05,P<0.01,P<0.001）.In the follow-up experiments,the concentration of IGF-1（25 ng/m L）was chosen.3.IGF-1 treatment alone could significantly induce the m RNA expressions of M2 phenotypes markers（Arg-1 and IL-10）in primary microglia.This result suggested that IGF-1could promote the microglia differentiation to M2 phenotype.4.Treatment alone with IGF-1 could increase the phosphorylation of Akt and ERK as well as the protein expression of GPER in a time-dependent manner（P<0.05,P<0.01）.IGF-1R specific blocker JB-1,phosphatidylinositol 3 kinase（PI3K）antagonist LY294002 or mitogen-activated protein kinase（MEK）antagonist PD98059 could inhibit IGF-1-induced protein phosphorylation of Akt or ERK（P<0.01）.JB-1,LY294002 and PD98059 could also inhibit IGF-1-induced protein up-regulation of GPER（P<0.001）.These results indicated that IGF-1 could promote the expression of GPER protein through IGF-1R-mediated signal pathway.5.To further clarify the inhibitory mechanism of IGF-1 on the inflammatory response of M1 microglia,BV2 microglia were pretreated with JB1 or G15.The results showed that JB-1 and G15 could block the inhibitory effect of IGF-1 against LPS-induced up-regulation of i NOS and COX-2 m RNA and protein expressions（P<0.05）.IGF-1treatment alone did not affected the m RNA and protein expression of the above pro-inflammatory factors.6.Lentivirus-mediated si RNA knockdown of GPER resulted in a significant decrease in the inhibitory effect of IGF-1 against LPS-induced m RNA expressions of i NOS、COX-2、TNF-αand IL-1βin BV2 microglial cells（P<0.01,P<0.001）.7.The primary microglia of GPER^+/+（wild type）and GPER^-/-（gene knockout）mice was used to establish the inflammatory cell model.The results showed that LPS could induce the m RNA expressions of i NOS,COX-2,TNF-αand IL-1β（P<0.001）.Meanwhile,the sensitivity of primary microglia of GPER^-/-mice to LPS was significantly increased compared to primary microglia of GPER^+/+mice.In primary microglia of GPER^+/+mice, IGF-1 significantly inhibited the inflammatory response induced by LPS（P<0.01）.While in primary microglia of GPER^-/-mice,the anti-inflammatory effect of IGF-1 was significantly decreased（P<0.01）.Conclusions:In summary,IGF-1 promotes the differentiation of microglia into immunosuppressive M2 phenotype and inhibits LPS-induced inflammatory response by activating IGF-1R signal pathway.Estrogen membrane receptor GPER gene deletion significantly inhibits the anti-inflammatory effect of IGF-1,suggesting that GPER is involved in the inhibitory effect of IGF-1 against LPS-induced microglial inflammation.