Study On ICA69 And Puerariae Decoctions In Pain Occurrence And Its Treatment

Pain is a kind of physiological and psychological complex clinical symptoms, and is also one of the most common symptom in clinical, and serious influence the patient’s quality of life. The occurrence of pain cause serious adverse effects on the patient’s physical and mental. The pain is defined as a kind of related to tissue damage or potential tissue damage unpleasant subjective feelings and emotional experience by the international association of pain.The reasons could lead to pain are exsit widely. So the effects of pain on the human health very extensive. The world health organization suggest that pain is the fifth life signs except breathing, blood pressure, body temperature and pulse. Due to these reasons, more and more sciencist pay attation to the research of pain, including drug development for the treatment of pain, neurophysio logical mechanism of pain. In the numerous research directions of the pain, the research of pathophysiology of pain is the most important.Along with the deepening of the research on mechanism of pain, a lot of important molecules were found associated with pain. Although in recent years, the results of pain mechanism have been reported, but the mechanism of pain caused by different pathogenesis and disease is quite different. The molecules which play an important role in these pain process is different. So the research of mechanism of pain is also an important work for sciencist. Meanwhile, the research related to mechanism of pain has very important value for clinical treatment of pain.Islet cells antigen protein 69 (ICA69) is composed of 480 amino acids, the molecular weight of 69 kD protein. The ICA69 was found in patients islet cells with type 1 diabetes. In recent years, ICA69 was found high expression in a variety of disease which could causes pain, such as diabetes neuropathy pain, rheumatoid arthritis, and the relationship between the expression of ICA69 and degree of pain was a positive correlation. The relationship between ICA69 and pain rare reported by scienceist. The aim of the research is explore the role of ICA69 in the pain, and provide basic research data for the reaserch of pain pathophysio logical.Chinese medicine treat disease for thousands of years, accumulated a lot of methods and experience of treatment of pain and provides more variety of effective treatment methods, including acupuncture and moxibustion, massage, gua sha, herb tea and so on. Puerariae decoctions is a famours Prescription, Clinical and basic research shows that the formula is effective for the treatment of diabetes, and it can significantly improve the primary symptom of diabetes. Because ICA69 is closely related to the occurrence of diabetes, and diabetes common complication after contains a continuous persistent spontaneous pain and allergy, so to observe the effect of puerariae decoctions on the pain induced by diabetes become another important content of this study.Experiment Objectives:explore the role of ICA69 in the pain which induced by different reasons. Preliminary experiments results show that the pain behavior of the mice was signifent decreased in formalin induced pain model when ICA69 was knocked out. The results note that ICA69 is required in inflammation pain. So to investigate the mechanism of ICA69 intervention inflammation pain become a meaningful work. The work carried out from two aspects, one is ICA69 in the role of inflammation, and the other is the role of ICA69 in peripheral nervous system. Eventually proved the role of ICA69 in inflammatory pain. To observe the effect of puerariae decoctions on the pain induced by diabetes.Experiment Method:(1) To observe the effects of ICA69 in different types of pain through behavioral experiments. (2) Application PCR technique to detect ICA69 expression in the nervous system. To observe the molecular properties of ICA69 positive neurons in dorsal root ganglia by immunohistochemical fluorescence staining. (3) Western blot is applied to detect expression level of ICA69, TRPV1, and CGRP in inflammation model group animal local inflammation tissue and dorsal root ganglion neurons. The level of TRPA1 was also detected by RT-PCR. (4) The release of inflammation skin tissue was extracted from WT and ICA69-/-mice respectively. Skin releases are used for calcium imaging experiments. According to the results of calcium imaging experiments, determine the role of ICA69 in inflammatory pain. (5) According to the results of experiment four, confirm that ICA69 could influence the inflammation process. In order to further explore the mechanism of ICA69 intervention inflammation, swelling of inflammation tissue and inflammatory cytokines (TNF-α, IL-1β and IL-6) were detected. Meanwhile, the activation of nuclear transcription factor NF-κB was detected and compared between WT mice and ICA69-/- mice. (6) Through single intraperitoneal high-dose STZ preparation of diabetes pain model in mice, the model was administered water extraction liquid of puerariae decoctions three weeks and then observe the animal model’s blood glucose, body weight, the influence of pain behavior, and further tests on the mice in the pancreas ICA69 expression level and serum levels of inflammatory cytokines.Experiment Results:(1) The pain induced by formalin could prevent through knockout ICA69. However, thermal pain and mechanical pain were not affected when lack of ICA69. (2) Most of ICA69 positive neurons are small diameter and unmyelinated peptide neurons. The expression of TRPV1, CGRP and TRPA1 had no significant change in the ICA69-/- mice. (3) ICA697-/- mice was used for inflammation pain model, the levels of ICA69, TRPV1, TRPA1, CGRP have no effected compared to control group animals.(4) the results of calcium imaging experiments show that the skin release from ICA69-/- mice is different to WT mice. The same neurons were cimulated by skin release from WT and ICA69-/- mice repectively, the release from WT mice could induce more neurons reaction. However, the neurons from WT and ICA69-/-mice were cimulated by the same skin release produce the same reaction. The results demonstrated that ICA69 take part in the inflammation process. (5) Compare to WT model animal, the swelling volum of inflammation local tissue in ICA69-/- mice model animal was significant decreased. The expression of TNF-a and IL-1β was also decreased in ICA69-/- ice model. Cell experiment proved that the activation of nuclear transcription factor NF-κB decreased significantly when ICA69 knockout. (6) Water extraction liquid of puerariae decoctions could significantly improve the model animal change in body weight and blood glucose caused by STZ injection. The pain behavior results shows that the decreased pain threshold was reversed by puerariae decoctions. The results of western blot and real time PCR show that puerariae decoctions could decrease the high expression of ICA69 in pancreatic tissue of model animals. The inflammatory cytokines TNF-a and IL-1β in serum was significantly reduced by puerariae decoctions.Experimental Conclusion:(1) ICA69 expressed in peripheral nervous system and immune cells. (2) The inflammation pain induced by formalin is required for ICA69. (3) ICA69 knockout could reduce the inflammation pain induced by formalin through decrease activation of NF-κB and inhibit cytokine generation. (4) Puerariae decoctions has signifiently effective on the pain induced by diabetes, and the mechanism of this treatment effect is by reduce the expression of ICA69 and inhibit the chronic inflammation in animal model.