| “Neuropathic pain” is defined as pain caused by a lesion or disease of the somatosensory nervous system by the International Association for the Study of Pain. The lesion or disease may affect the peripheral nervous system(peripheral neuropathic pain) or the central nervous system(central neuropathic pain). Multiple types of tissue injuries result in neuropathic pain, such as trauma, infections, inflammation, chemotherapy, tumor infiltration, metabolic abnormalities and radiotherapy. The clinical findings in neuropathic pain have been summarized as allodynia(pain from a non-noxious stimulus), hyperalgesia(the lowering of pain threshold and an exaggerated response to a painful stimulus) and spontaneous pain. Neuropathic pain research becomes difficulty and study hot spot, because of the complication of pathogenesis and lacking of effective clinical control methods.Hepatocyte growth factor(HGF) is known to a multi-functional growth factor, including promotion of cell survival, differentiation, proliferation, angiogenesis, neurotrophin, anti-inflammation, and anti-fibrosis. However, the serum half-life of HGF protein is only 10 minutes, and it has a lot of limitations as a drug application. In order to overcome this disadvantage, we constructed the eukaryotic expression plasmid DNA carrying the kan+ resistance gene, cmv promoter, human hgf gene and ploy A(Named as p UDK-HGF, patent number ZL00132196.X). Currently, the phase I of clinical trial has been finished for treatment of peripheral arterial disease using p UDKHGF injection, and the phase II of clinical trials are being carried out in China. Phase I clinical results have shown that most obvious and early effect of local intramuscular injection p UDK-HGF is relief of pain caused by limb ischemia, ulcers, gangrene in CLI patients, this analgesic effect may be prior to angiogenesis. The results give a suggestion that the analgesic effect of p UDK-HGF on skin, muscle and nerve injuries induced neuropathic pain, but the mechanism is still unclear. Therefore, we study the analgesic effect and mechanism of p UDK-HGF on three different types of pain model in rodents, including plantar incision pain model, skin/muscle incision traction pain model and spared nerve injury model.Firstly, we established a production process of p UDK-HGF including a 50-liter fed-batch fermentation, continuous alkaline lysis, integrated three-step chromatography on Sepharose 6 Fast Flow, Plasmid Select Xtra, and Source 15 Q, and concentration and formulation. This process can be completed within one week resulting in pharmaceutical-grade plasmid DNA. The final p UDK-HGF product can be directly used for cell transfection and animal experiments. The HGF expression of transfected CHO cells was assayed at 40.5±2.2 ng/m L/1×106cell using human HGF ELISA kit after 48 h transfection. The ability of HUVEC to migrate through transwell filters was improved three times by cell treatment with supernatant of p UDK-HGF transfected CHO cells, compared with supernatant of control vector p UDK transfected CHO cells, with significant difference P < 0.01.Secondly, plantar incision pain model, skin/muscle incision traction(SMIR) pain model and spared nerve injury(SNI) model were used to research the analgesic effect of intramuscular or intrathecal administration of p UDK-HGF. The results showed that the spontaneous pain behavior, mechanical withdrawal threshold and withdrawal thermal latency were significantly improved at 6h after local intramuscular injection of p UDK-HGF in incisional pain rats. Meanwhile, the mechanical withdrawal threshold was significantly improved by local intramuscular or intrathecal injection of p UDKHGF in SMIR pain rats, suggesting that central sensitization was inhibited by p UDKHGF. In the SNI pain model, the mechanical and thermal withdrawal thresholds were improved by intrathecal injection of p UDK-HGF. Furthermore, we found the analgesic effect can prolonged up to 52 days after triplicate intramuscular or intarthecal injection of p UDK-HGF. In summary, the results indicate that p UDK-HGF prevents the neuropathic pain induced by skin, muscle and nerve injury.Thirdly, p UDK-HGF inhibited the infiltration of inflammatory cells, reduced collagen fiber accumulation, and activated muscle satellite cells proliferation to promote the injured muscle fibers repair and regeneration. The blood perfusion was measured using Doppler blood flow detector in the injured muscle tissues induced by SMIR or SNI. The results showed that the blood perfusion of injured muscle was improved by local intramuscular injection of p UDK-HGF, however, the blood perfusion out of the injection site did not change. We detected algogenic substance expressions for PGE2, 5-HT and HIS in injured muscle samples at a given defined time point in three groups(na?ve, p UDK and p UDK-HGF groups) of SMIR. Compared to na?ve group, the level of PGE2, 5-HT and HIS significantly increased and persisted at a high level in rats with p UDK treatment. However, the concentration of PGE2, 5-HT and HIS in the wound was found to be significantly decreased in p UDK-HGF gene transfer rats. Meanwhile, p UDK-HGF inhibited NGF expression in injured muscle, which possibly contribute to the attenuation of persistent postoperative pain. Furthermore, local intramuscular injection of p UDK-HGF decreased CD11 b, GFAP, TLR4 and TNF-α expressions in the lumbar segment L3-L5 of spinal cord, suggesting that spinal glial cells activation and pro-inflammatory factor expression were inhibited due to wound repair and inflammatory factor decreasing in injured muscle after intramuscular injection of p UDK-HGF. Above all, the peripheral analgesic mechanism of the p UDKHGF includes:(1) inhibition of inflammatory cell infiltration and activation of muscle satellite cells to promote the repair of tissue injury;(2) increasing local tissue blood perfusion to improve micro-environment of tissue damage;(3) inhibition of algogenic substance expression in injured tissues, and inhibition of peripheral pain nociceptor sensitization and excitement, suppression of peripheral sensitization;(4) inhibition of pain signals deliver to the spinal cord, reducing the spinal glial cells activation, inhibiting spinal cord inflammatory factor expression, and inhibiting the formation of central sensitization.Fourthly, the lumbar segment L3-5 of spinal cord was used for detection of glial cells activation on 7, 14, 32 days after intrathecal injection of p UDK-HGF in SMIR rats. The results showed that the CD11 b and GFAP expression in spinal cord were significantly decreased in p UDK-HGF treated SMIR rats, compared to rats with p UDK treatment. In vitro, rh-HGF protein inhibited BV2 cells morphological changes induced by LPS, inhibited inflammatory cytokines IL-1β, IL-6, TNF-α m RNA upregulation, and inhibited the i NOS m RNA upregulation and reduced the level of NO in cell culture supernatant. Taken together, the central analgesic mechanism of p UDK-HGF contains:(1) HGF protein is expressed in spinal cord dorsal after intrathecal injection of p UDKHGF, and inhibited the spinal microglia and astrocytes activation;(2) rh-HGF protein inhibits the microglia activation due to inhibit microglia morphological changes and Iba1 expression;(3) HGF protein inhibits the expression of inflammatory factor IL-1β, IL-6, TNF-α and NO in activated microglia, and inhibits the formation of central hypersensitivity.In conclusion, these results suggest that p UDK-HGF prevents the neuropathic pain induced by skin, muscle and nerve injury. Furthermore, our results provide a potential therapeutic strategy for refractory pain including postoperative persistent pain, chronic wound persistent pain, and sciatica pain. |
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