Mechanism Study Of The Anti-inflammatory Effect Of Icaritin Via ER And IGF-1R On Primary Astrocytes

Astrocytes(AC)are the most widely distributed and largest type of cells in the central nervous system and participate in a variety of physiological or pathological processes.Under physiological conditions,AC play an important role in energy metabolism regulation,neurotrophic factor secretion,glutamic acid homeostasis,neurotransmitter clearance,synaptic plasticity,and maintaining the integrity of the blood-brain barrier.A large number of studies have shown that reactive astrogliosis and astrocyte-mediated neuroinflammation are involved in the pathogenesis of neurodegenerative diseases,such as Parkinson's disease(PD)and Alzheimer's disease(AD).When the body is stimulated by injury,microglia are first activated,astrocytes can amplify microglia-derived inflammatory response.Eventually,it promotes neuronal damage and degeneration.Therefore,inhibiting the inflammatory response and alleviating the over-activation of astrocytes will be a promising therapeutic target for the treatment of neurodegenerative diseases.Herba Epimedii is a traditional Chinese medicine,which belongs to the genus epimedium.Total flavonoid of Herba Epimedii is the major active components which process a variety of biological activities including tonify kidney,anti-osteoporosis,anti-aging,immunity improvement,tumor inhibition,and anti-inflammation.Icaritin(ICT),the main active ingredient of Epimedium,has an estrogen-like steroid skeleton structure.Studies have shown that ICT could against ? amyloid-induced neurotoxicity via estrogen nuclear receptor(estrogen receptor?/?,ER?/?)-dependent pathway to exert the neuroprotective effect in primary rat neuronal cells.Our previous studies have demonstrated that G protein-coupled estrogen receptor(GPER)and insulin-like growth factor-1 receptor(IGF-1R)signaling pathways were involved in the anti-inflammatory effect of ICT in BV2 microglia cell.However,whether ICT can inhibit the inflammatory response of primary astrocytes through GPER,ER?/? and IGF-1R remains unclear.In the present study,real time PCR and western blot technique were used to study the anti-inflammtory effects of ICT and to confirm the involvement of GPER,ER?/? and IGF-1R using pharmalogical blockade in lipopolysaccharide(LPS)-induced rat ventral mesencephalic primary astrocytes inflammatory model.In addition,in order to further clarify the mechanism of ICT,we also explored the effects of ICT on astrocytes physiological function and the possible signaling pathways.The experimental results are as follows:1.Mechanism study of the inhibitory effects of icaritin against LPS-induced astrocytes inflammatory response.(1)LPS(1 ?g/m L)treatment significantly increased gene expressions of tumor necrosis factor ?(TNF-?)and interleukin-1?(IL-1?)(p<0.001)in primary astrocytes.Different concentrations of ICT(0.1,1,10 and 20 ?M)could dose-dependently inhibit LPS-induced inflammation.The most significant effect occurs at 10 ?M and 20 ?M(p<0.001).We used 10 ?M in subsequent experiments.(2)GPER antagonist G15(1 ?M)could block the inhibitory effect of ICT on LPS-induced mRNA expressions of TNF-? and IL-1?,cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(i NOS)(p<0.05,p<0.01).(3)G15 could not block the inhibitory effects of ICT on LPS-induced COX-2 and i NOS protein expressions(p>0.05).(4)ER?/? antagonist ICI182,780(1 ?M)could not block the inhibitory effects of ICT on LPS-induced mRNA expressions of TNF-?,IL-1?(p>0.05),but could block the inhibition of mRNA expressions of COX-2 and i NOS(p<0.01,p<0.05).(5)IGF-1R antagonist JB-1 could block the anti-inflammatory effects of ICT on mRNA expressions of TNF-?,IL-1?,COX-2 and i NOS(p<0.001).(6)Both JB-1 and ICI182,780 could block the inhibitory effects of ICT on LPS-induced COX-2 and i NOS protein expressions(p<0.05,p<0.01,p<0.001).2.Mechanism study of the regulatory effects of ICT on physiological function of astrocytes.(1)ICT treatment alone(1,2,4 and 6 h)could up-regulate the mRNA expressions of glial cell-derived neurotrophic factor(GDNF)and c-fos in a time-dependent manner.GDNF was up-regulated most significantly at 2 h(p<0.001),and the ion of c-fos was up-regulated most significantly at 1 h(p<0.001).(2)Both G15 and JB-1 pretreatment could block the regulatory effects of ICT on the gene expressions of GDNF and c-fos(p<0.05,p< 0.01).(3)ICT treatment alone(1,2,4 and 6 h)could up-regulate the mRNA expressions of glutamate transptor-1(GLT-1)and glutamate-aspartate transporter(GLAST)in a time-dependent manner(p<0.05,p<0.01).(4)Both G15 and JB-1 could block the regulatory effects of ICT on the mRNA expressions of GLT-1 and GLAST(p<0.05,p<0.01).(5)ICT treatment alone(5,10,20 and 30 min)could remarkably increase the phosphorylation level of c AMP responsive element-binding protein(CREB)(p<0.01),and this effect could be blocked by G15(p<0.05).(6)Exposure to ICT for 5,10,20 and 30 min,the phosphorylation levels of ERK and Akt significantly increased in a time dependent pattern.The maximum occurred at 30 min(p<0.01).(7)JB-1 could block the ICT-induced phosphorylation of ERK and Akt(p<0.05).Conclusion: Taken together,ICT can inhibit LPS-induced inflammatory response in ventral mesencephalic primary astrocytes.Its anti-inflammatory mechanism may be related to GPER,ER?/? and IGF-1R signaling pathways.ICT can regulate the physiological function of astrocytes via GPER and IGF-1R.This study provides direct experimental data for the development of traditional Chinese medicine.