The Research Of Neuregulin-1Mediating Astrocytes Activation In The Development Of Chronic Neuropathic Pain

ObjectThe aim of this study is to research the relationship between HRG and astrocytes thoughobserving the effect of exogenous NRG1(HRG) on the proliferation and chemotaxis ofastrocytes cultured in vitro. To analysis the effect of neuregulin-1(NRG1) mediatingastrocytes activation in the development of chronic neuropathic pain (NPP) from theexperiment of observing the effect of HRG on the basis pain threshold by giving HRG tothe male SD rats though intrathecal catheterization and the experiment of observing thebehavior changes and detecting the activation of astrocytes on the level of spinal cord afterpretreated with lentivirus NRG-siRNA before spinal nerve ligation (SNL) model.Methods1. The effect of HRG on the proliferation and chemotaxis of astrocytes cultured invitro:Primary culture astrocytes in vitro from bilateral cerebral cortex obtained from SD ratsborn in3days. After the cells were confluent on the bottom of the culture flask,37℃200rpm20h in orbital shaker so that to reduce microglial cells and fibroblasts. Thenastrocytes of the purity95%were chose for the next experiments.1.1The effect of HRG on the proliferation of astrocytes cultured in vitro1.1.1The effect of different doses on the proliferation of astrocytes cultured in vitroThe astrocytes was passaged and plated in24wells plates at a density of4×104cells/mL, then randomly divided into5groups(n=3) and given drugs so that the final separateconcentrations were: HRG0nM, HRG1nM, HRG5nM, HRG10nM, HRG20nM. The24well plates were put in cell incubator, when the cells reached the logarithmic phase (on theday of3), EdU was added to the medium (1:2000), and the method of EdU-incorporationwas used to detect the effect of HRG on the proliferation of astrocytes.1.1.2The analysis of the receptors pathway about the effect of HRG on the proliferation ofastrocytes cultured in vitroThe cells cultured and purified according to the above methods were plated in24wellplates at a density of4×104cells/mL, then randomly divided into4groups(n=3) and givendrugs so that the final separate concentrations were: NS2μL/mL, HRG10nM, HRG10nM+AG147810μM, HRG10nM+AG87910μM. When the cells reached the logarithmicphase (on the day of3), EdU was added to the medium (1:2000), the method ofEdU-incorporation was used to detect the proliferation of astrocytes.1.2The effect of HRG on the chemotaxis of astrocytes cultured in vitro1.2.1The effect of different doses on the chemotaxis of astrocytes cultured in vitroThe cells cultured and purified according to the above methods were plated intranswells and randomly divided into5groups (n=3), then analysis the effect of HRG onthe chemotaxis of astrocytes cultured in vitro. Freshly prepared astrocytes suspension(serum-free DMEM/F12,4000cells/100uL) were placed into the top wells of transwells,the bottom wells of transwells were filled with600uL serum-free DMEM/F12or NRG1ofvarious concentrations so that the final separate concentrations were: HRG0nM, HRG1nM,HRG5nM, HRG10nM, HRG20nM. The transwells were kept in incubator (37℃,5%CO2,100%humadity). After6hours, the cells above the membranes were removed and the cellsunder the membranes were counted by microscope after the staining of DAPI. The cellsunder the membranes were considered the chemotactic astrocytes.1.2.2The analysis of the receptors pathway about the effect of HRG on the chemotaxis ofastrocytes cultured in vitro The cells cultured and purified according to the above methods were plated intranswells and randomly divided into4groups (n=3), then analysis the effect of HRG onthe chemotaxis of astrocytes cultured in vitro. Freshly prepared astrocytes suspension(serum-free DMEM/F12,4000cells/100μL) were placed into the top wells of transwells,the bottom wells of transwell were filled with600μL serum-free DMEM/F12or HRG ofvarious concentrations so that the final separate concentrations were: NS2μL/mL, HRG10nM, HRG10nM+AG147810μM, HRG10nM+AG87910μM. The transwells were keptin incubator (37℃,5%CO2,100%humadity). After6hours, the cells above the membraneswere removed and the cells under the membranes were counted by microscope after thestaining of DAPI. The cells under the membranes were considered the chemotacticastrocytes.2. The effect of NRG1on the development of chronic neuropathic pain2.1The effect of HRG on the basis pain threshold of rats32male SD rats were randomly divided into4groups (n=8): control (normal saline20μL), HRG (10ng), HRG(10ng)+AG1478(10μg), HRG(10ng)+AG879(10μg). PE-10wasinserted the catheter via the foramen magnum and the head reached the lumbarintumescentia of spinal cord. When the intrathecal catheterization was succeed, the drugswere deliveried though PE-10according to the groups. The behavior changes were detectedbefore given drugs every day. At the day of14after given drugs, the rats were anesthetizedand the lumbar intumescentia of spinal cord was removed, then the activation of astrocytesin the cornu dorsale medullae spinalis was observed though immunofluorescence staining.2.2The effect of NRGl down regulated by lentivirus NRG1-siRNA on the developmentof chronic neuropathic pain32male SD rats were randomly divided into4groups (n=8): Sham+control lentivirus,Sham+lentivirus (107TU), SNL+control lentivirus, SNL+lentivirus (107TU). PE-10wasinserted the catheter via the foramen magnum and the head reached the lumbarintumescentia of spinal cord. When the intrathecal catheterization was succeed, the lentivirus were deliveried though PE-10according to the groups. After one week, the SNLmodel was made. The behavior changes were detected on alternate days after operation. Atthe day of14after operation, the rats were anesthetized and the lumbar intumescentia ofspinal cord was removed, then the activation of astrocytes in the cornu dorsale medullaespinalis was observed though immunofluorescence staining.Results1①HRG was given to the astrocytes cultured in vitro, compared with the group ofcontrol, the proliferation of astrocytes of HRG group was increased obviously (P<0.05).However, when the receptor-antagonists were given to the astrocytes at the same time withHRG, the astrocytes were reduced to50%compared with control (P<0.05). These resultsdemonstrated that HRG can dose-dependently promote the proliferation of astrocytescultured in vitro and the effect is mediated by the receptor of ErbB2/4.②HRG was givento the astrocytes of transwells cultured in vitro, compared with the group of control, theastrocytes that chemotax under the side of membranes of HRG group was increasedobviously (P<0.05). However, when the receptor-antagonists were given at the same timewith HRG, the astrocytes that chemotax under the side of membranes were reduced2/3compared with the group of HRG (P<0.05). These results demonstrated that HRGdose-dependently promote the chemotaxis of astrocytes cultured in vitro and the effect ismediated by the receptors of ErbB2/4.2①When given HRG, the male SD rats appeared the behavior changes like chronicneuropathic pain, compared with the group of control, the threshold of mechanical pain andthe thermal pain were decline (P<0.05) and the astrocytes of spinal cord were activated.However, when the receptor-antagonists were given at the same time with HRG, the aboveeffects were inhabited, compared with the group of HRG, the threshold of mechanical painand the thermal pain of the group of AG1478or the group of AG879were up-regulated.The results demonstrated that NRG1was a pain-producing factor at the level of spinal cord,and may play a role though activated the receptors ErbB2/4of astrocytes.②After the expression of NRG1of the level of spinal cord was down-regulated by lentivirus, thedecreased of pain threshold and the activation of astrocytes were put off. At the same timeafter SNL, compared with the group of SNL+control lentivirus, the pain threshold ofgroup of lentivirus was up-regulated (P<0.05); the results of immunofluorescence stainingdemonstrated that at the day of14after operation, compared with the group of SNL+control lentivirus, the activation of astrocytes of spinal cord were inhibited. Above theseresults demonstrated that NRG1play an important part in the development of neuropathicpain after SNL, this may mediated by the activation of astrocytes though the receptor ErbBof astrocytes.Conclusions①The receptor of ErbB was expressed on the astrocytes cultured in vitro. HRG candose-dependently promote the proliferation and chemotaxis of astrocytes cultured in vitroand the effect is mediated by the receptor of ErbB2/4of astrocytes. These were providedtheoretical basis for the research of NRG1activating astrocytes contributed to theneuropathic pain.②The relationship between NRG1and the activated astrocytes in thedevelopment of neuropathic pain was certified and elaborated first.③After SNL model,the rats’ thresholds of mechanical and thermal pain were declined and the astrocytes ofspinal cord were activated but after NRG1of the level of spinal cord was down-regulatedby lentivirus, the decreased of pain threshold and the activation of astrocytes were put offwhich demonstrated that astrocytes activation mediated by NRG1play an important part inthe development of neuropathic pain.