The Mechanisms On The Enhanced Excitability Of Primary Sensory Neurons By Lysophosphatidic Acid In A Model Of Cholestatic Itch

Chronic pruritus is a common and unbearable clinical symptom, which is often accompanied by the occurrence of a variety of systemic diseases, such as: chronic hepatobiliary disease, chronic renal failure, cancer, systemic infection, endocrine disorders and blood diseases. Recently, as people’s living standards improve, people’s attention on chronic pruritus-related fields has been increasing. In recent years, some specific neural pathways have been discovered, including some new and their corresponding receptors, however, the specific pathogenesis of chronic pruritus is still not very clear, and an effective treatment has not been found.In recent years, lipid metabolism dysfunction is a hot clinical research key point. LPA(lysophosphatidic acid, LPA) in lipid metabolism, as an important intermediate product, it is becoming more and more attractive, which is mainly composed of platelets, fibers, fat cells and secretory cells in a variety of diseases, through a variety of signal transduction pathway and plays an important biological role. Recently, Kremer and other studies have found a new type of itch-transmitter induced lysophosphatidic acid(LPA) in serum of chronic cholestatic pruritus patients, which has been proved to be abnormally elevated in chronic cholestatic pruritus caused by important factors. Studies have indicated that there is a class, specifically expressed in mouse dorsal root ganglion neurons of mast cell-associated G protein-coupled receptors A3(Mas-Related G-coupled Protein Receptor, MRGPRA3) and chemically closely related pruritus. MRGPRA3 positive neurons are closely associated with conventional histamine-dependent itching behavior. Although such nociceptors play a role in mediating acute itching irritation, but it remains unclear in chronic cholestatic pruritus pathological conditions, such as the change of neuronal excitability. After the above reasoning, we can assume that, in cholestatic pruritus, LPA may activate the itch-specific MRGPRA3 positive neurons, through binding a particular type of LPA receptor, then the relatively increase neuronal excitability produce spontaneous scratching behavior.After the implementation of this study, we firstly use the male C57 mice to establish common bile duct ligation model(BDL surgery), to detect the number of spontaneous scratching behavior at the different days after the mice with BDL surgery. Then, primary cultured dorsal root ganglion(Dorsal root ganglion, DRG) neurons were stimulated by the administration of LPA. Then we observed the changes of intracellular calcium ion concentration and the alterations of cell membrane excitability within the small-diameter DRG neurons. Next, the researchers investigated the specific sub-type receptor of LPA-induced itch by molecular biology methods, and using the knockout mice to further confirm the interaction with the LPA receptor. Finally, we used the MRGPRA3 transgenic mice by in vivo electrophysiological experiments method, to clarify the clear mechanism between the LPA induced itch and the MRGPRA3 positive neurons. The results are as follows: 1. The increase of spontaneous scratching numbers in cholestatic miceEthology results show that with the time extension of the common bile duct ligation, compared with the control group of mice, cholestasis bile duct ligation in mice in the first 10 days after surgery spontaneous itch scratching behavior within one hour induced by cholestasis peaked and then began to decline, and the observation was ended by day 14.2. LPA increase calcium levels and cell membrane excitability in small diameter neuronsRespectively, primary cultured DRG neurons of mice from sham group and BDL group, using calcium imaging techniques(calcium ratiometric imaging), were compared after LPA stimulation, different model groups of small diameter DRG neurons calcium differences LPA reactive changes, we found that compared to intracellular small diameter DRG nerves in sham-operated mice, the small diameter DRG positive neurons cells in common bile duct ligation group caused higher percentage increase in calcium response amplitude, with significant statistical significance.Moreover, percentage of small diameter positive DRG neurons cells in sham group of mice, responded to the LPA, is about 21.2%, on the contrary, for a small-diameter DRG neurons in the common bile duct ligation group of mice, which respond to LPA, the percentage of positive cells significantly increased, reaching about 46.2%, with significant statistical significance.These results can be explained, as compared with the sham group, LPA can cause the increase of the ratio of LPA induced positive responsive cells calcium reaction in the mouse small-diameter DRG nociceptive sensory neurons from the common bile duct ligation group, and the increase is partly due to the move of calcium from extracellular calcium into the intracellular calcium.Topical LPA enables resting membrane potential(RMP) of small diameter DRG neurons to cause significant discharge, with significant statistical significance. Moreover, membrane depolarization can make these responsive neurons evoke action potentials(AP) discharge. During the processing of the LPA, the average input resistance(Rin) significantly decreased, the strength of the base significantly decreased, the base strength evoked by 2 times the number of action potentials increased, with statistically significance, indicating that open of ion channels in resting increased. These results suggest that, LPA can directly activate primary sensory neurons in the bile duct ligation group. 3. LPA increased scratching behavior by LPA4 receptors, intracellular calcium levels and cell membrane excitabilityTo detect different subtypes of the lysophosphatidic acid receptor(LPAR1-6) expression levels in the spinal cord dorsal root ganglion at the bile duct ligation mice, we used Real-time PCR method. From the PCR test results, we found that compared with sham-operated mice, LPAR4 receptor mRNA expression levels were significantly increased at day 10 after BDL surgery, with statistically significance, however, LPAR1-3,5,6 receptor does not keep statistics differences between sham and BDL groups.For LPAR4 knockout mice, subjected to the same common bile duct ligation operation, compared with the wild-type mice, from day 10 to the day 14 after the common bile duct ligation, LPAR4 knockout mice significantly reduced the counts of spontaneous itching scratching behavior. Topical LPA stimulation to the LPA4- /- small diameter neurons, compared with the sham group, the percentage of DRG neurons with little positive response to LPA significantly reduced in the bile duct ligation LPA4- /- mice, with statistically significance. Moreover, compared with wild mice, the number of positive small neurons cells and the rate of increase induced by LPA in the LPA4- /- group were significantly weakened. We also found that, compared to wild type mice, LPA causes resting membrane potential depolarization significantly decreased in the LPA4 knockout mice, the average input resistance(Rin) increased, the strength of the base increased, the number of action potential evoked by twice of the fundamental strength reduced. 4. LPA mediated by LPA4 MRGPRA3 itch neurons participating cholestasis pruritusUsing MrgprA3 transgenic mice, topical application of LPA can cause primary cultured MrgprA3 + positive neurons within the calcium ions reaction significantly increased in the bile duct ligation model group. By in vivo electrophysiological techniques, topical application of LPA can cause increased Mrgpr A3 + neuronal excitability in the BDL group.In summary, through this research, we found that increasing cholestatic mice spontaneously itch scratching times. At the same time, we found that with LPA4 at LPA receptor-specific binding site, the number of positive cells in the neuronal intracellular calcium ions react, and topical use can increase the magnitude of response and cell membrane excitability, LPA-induced cholestatic pruritus is partly due to the participation of chloroquine, a non-histamine-dependent itching, and "the use of antihistamines to treat cholestatic pruritus invalid" on the clinical point of view remained the same. Moreover, we also found that LPA receptor mediated by LPA4 in the MRGPRA3 itch neurons participate in cholestasis pruritus.