Effects Of Substance P On Astrocyte Activation And The Underlying Mechanisms

Objective:1.Explore the implication of substance P in activation of astrocytes in vitro,concerning on cellular morphological changes and the level of IL-1β and IL-10 released.2.Determine whether and how the concentration of intracellular calcium is affected after astrocytes are treated with substance P.Methods:1.Primary cultures of astrocytes were prepared from SD rats born in 0-1 day.Astrocytes harvested during the 10 th to 12 th day have been divided into 5 groups:(1)control group, incubated with complete medium;(2) complete medium mixed with substance P 30 n M;(3) complete medium mixed with substance P 100 n M;(4)complete medium mixed with substance P 300 n M;(5) complete medium mixed with substance P 1000 n M. Cell supernatants were separately collected at 2 h, 6 h, 12 h and24 h after incubation. Simultaneously cells were reserved for further studies.2.Astrocytes were identified with GFAP, the morphological changes of astrocytes incubated with substance P in different concentrations were observed by immunofluorescence assay.3.The release of IL-1β and IL-10 from astrocytes incubated with substance P were measured by enzyme-linked immunosorbent assay.4.Changes of intracellular calcium in astrocytes treated with substance P was recorded by Meta Flour of the high-content imaging system.Results:1.Indentification of astrocytes in vitro: Astrocytes were confirmed with GFAP antibody by immunofluorescence assay. The purity of cultured astrocytes reached95% according to restaining result of DAPI.2.Morphological changes of astrocytes incubated with substance P: Astrocytesincubated with cell medium as control, astrocytes incubated with substance P 30 n M for 2 h、6 h、12 h or 24 h showed no morphological changes. After incubated with substance P 100 n M, somatas of some astrocytes began to grow bigger and round and simultaneously processes of some astrocytes retracted, the number of morphological changed cells increased and fluoresence intensity of GFAP enhanced over time. The effects of substance P 300 n M on astrocytes were more higher than substance P 100 n M. Astrocytes incubated with substance P 1000 n M for 2 h start to show morphological changes, but fluoresence intensity of GFAP is weaker after 6 h and 12 h than groups incubated with substance P 100 n M or 300 n M. Fluoresence intensity of GFAP of astrocytes incubated with substance P 100 n M or 300 n M at 24 h become weaker than 12 h.3.Elisa assay results of IL-1β: Astrocyte supernatant incubated with complete medium as control group. There is no difference between group incubated with substance P 30 n M at 2 h、6 h or 24 h with control group. But the level of IL-1β from group incubated with substance P 30 n M for 12 h is higher than control group. The level of IL-1β from group incubated with substance P 100 n M or 300 n M for 2 h、6 h and 12 h is also higher than control group. But there is no difference between group incubated with substance P 100 n M or 300 n M at 24 h with control group. It indicates that substance P may potentiate IL-1β release from astrocytes, and the potentiation have a specific time process. This potentiation is especially obvious after incubation with substance P 30 n M for 12 h, and disappeared at 24 h. Potentiation of groups incubated with substance P 100 n M or 300 n M starts at 2 h, becomes obvious at 6 h.With time, the potentiation weakens and disappears at 24 h.4.Elisa assay results of IL-10: Astrocyte supernatant incubated with complete medium serve as control group. The level of IL-10 from groups incubated with substance P 30 n M、100 n M or 300 n M for 2 h or 6 h decrease. There is no difference between groups incubated with substance P in 30 n M、100 n M or 300 n M for 12 h with control group. The level of IL-10 from groups incubated with substance P 30 n M、100 n M or 300 n M for 24 h increase, while no significance can be seen after astrocytes incubated with substance P separately 100 n M、300 n M or 1000 n M for 12 h. It suggests that substance P may inhibit IL-10 release after incubated for 2 h and 6h. With time, the inhibition weakened and release of IL-10 increases.5.Intracellular calcium imaging results: Astrocytes incubation with Hanks buffersolution are taken as control group. kcl of 60 m M is used to confirm functional activity of astrocytes. Intracellular calcium from groups treated with substance P 30 n M、100 n M、300 n M or 1000 n M increase, contrast with control group. Intracellular calcium from group treated with substance P 100 n M is higher than groups treated with substance P 30 n M 、 300 n M or 1000 n M. Intracellular calcium from group treated with substance P 300 n M is more than groups treated with substance P 30 n M or 1000 n M. There is no difference between group incubated with substance P 30 n M and group incubated with substance P 1000 n M. It illustrates substance P increases intracellular calcium in astrocytes.Conclusions:1.Substance P can activate astrocytes, exhibiting morphological changes.2.Substance P can potentiate IL-1β release and inhibit IL-10 release from astrocytes.3.Activation of astrocytes by substance P can persist for 12 h, weakened at 24 h.4.Substance P may increase intracellular calcium of astrocytes.5.Substance P may be involved in pain by activating astrocytes, including increase intracellular calcium, potentiation of IL-1β release and inhibition of IL-10 release.Chronic neuropathic pain is produced by injury to the somatosensory nervous system, characterized by abnormal sensations such as allodynia(pain produced by normally non-painful stimuli) and hyperalgesia(excessive pain from noxious stimuli).Chronic pain can persist for several months and can be very difficult to manage or treat. Research on pain in the past decades has made great achievements. The different central areas of pain, their function and association, local neuronal circuits, working mechanism of nociceptor and neurochemicals involved in pain transmission have been further understood. However, previous studies focused on neurons, their circuits and effects of neurotransmitters. Drugs based on these findings are designed to target neurons in pain transmission, but treatment for chronic pain is not satisfactory. Glia play an important role in the pain signal generation, transmission and formation. They are gradually considered to be attached to research and treatment of pain and become an important target.Glia in the nervous system, are far more than neurons. More and more dataindicate role of glia under neurophysiological and neuropathological conditions is very important. In animal models of pain associated with chronic pain symptoms,simultaneously are activated glia in the spinal cord, exhibiting the mast cell body,dendrites retraction, increased expression of surface markers and so on. And intrathecal or systemic application of glial metabolic inhibitors, inhibiting activation of spinal glia, can relieve symptoms of chronic pain. These results confirm that activation of glia in the spinal cord is associated with chronic pain. But because the nervous system is a complex loop of neurons and glia, conventional pain model can not be used for in-depth, systematic observation on glia, and conclusions are difficult to exclude the impact of neurons. Therefore the role and mechanism of glia in pain signal generation and transmission, such as how glia are activated, how they work after activation are not clear.Rats of pain model, pretreated with lidocaine at injured site, expression of GFAP was decreased. And GFAP is specific marker protein of astrocytes. These results show that activation of glia in the central nervous system by noxious stimuli depends on neurons. It indicates nociceptive signals generated from neurons, and then transmitted toward spinal cord may start astrocyte activation..The material basis of sensory signals transmit from peripheral sensory neurons to spinal cord is the neurotransmitters. Substance P, a member of the tachykinin peptide family, is pain specified neurotransmitter in spinal cord. It has the highest affinity to NK-1 receptor, which is also widely distributed on membrane of glia other than neurons. The conventional experiment results in vivo are obtained on the basis of neuronal and glial network, which cannot tell clearly about direct effect of substance P on astrocytes. That is, whether and by what pathway substance P will have a direct stimulation to activate astrocytes, whether astrocytes are involved in pain transmission and regulation through the synthesis and release of cytokines(including inflammatory cytokines and anti-inflammatory cytokines). Therefore, our study will observe activation of astrocytes by pain-specific neurotransmitter substance P in vitro and explore related mechanisms.