Efficacy Assessment And Molecular Mechanism Of Yuanhu Zhitong Prescription For Treating Neuropathic Pain

BackgroundYuanhu Zhitong tablets have efficacy of qi and blood activation and pain relief, which can be applied with remarkable effect for pain with qi stagnation and blood stasis. Active ingredient groups have been gained through qualitative and quantitative analysis of constituents, ADMET analysis and the active evaluation of the intestinal absorption fluid on vaso-activity. And it is interesting that several ingredients can target pain, anxiety and depression by network pharmacology. However, pharmacodynamic properties and molecular mechanism have not been clear.Chronic pain can be divided into kinds of types. Among them, neuropathic pain is difficult to cure, which can be caused by injury of somatosensory system or diseases, and affect over 5% population all over the world. In recent years, several components have been identified from Rhizoma Corydalis yanhusuo and Radix Angelica Dahuricae to attenuate neuropathic pain without tolerance.Taking the above into consideration, the study is designed to investigate whether Yuanhu Zhitong prescription could relieve neuropathic pain in CCI rats and to explore the underling molecular mechanisms, which will lay foundations for the new indication in clinic and drug development.MethodsThis research is divided into 3 parts, involving animal model establishment, pain relief assessment and molecular mechanism.Part One, CCI model establishment4 ligatures were tied loosely around the left sciatic nerve. Mechanial hypersensitivity and thermal allodynia were used to confirm the stability of the model. Meanwhile, gabapentin, a common drug in clinic, as a kind of postive drugs was used to determine the successful establishment of chronic constriction injury model.Part Two, pain relief assessment of YZPAfter CCI, the extraction of YZP with different doses, with gabapentin (gab) as the first positive drug and Lutongding (LTD) as the second positive drug were administered orally for two weeks. Mechanical hypersensitivity and thermal allodynia were assessed by measuring rats on the 1st,3rd,7th,14th,16th,18th, and 21st after surgery during the period of infusion.Part Three, molecular mechanism of YZP for neuropathic painThe cerebrospinal fluid (CFS) and L4-L6 dorsal roots (DRG) were harvested on the tenth day of postoperation. CFS was detected for the quantify of four kinds of amino acid, which could act as biomarkers for clinical examination. L4-L6 DRGs were extracted for total RNA and applied for miRNA and gene microarray technology, respectively. Different expression of miRNAs and mRNAs were gained. MiRNAs were predicted with Targetscan and miRTarBase programs for their targeting mRNAs, which were then intersected with different expression of mRNAs.And then candidate miRNAs and their targeting mRNAs selected in the therapies of YZP for neuropathic pain were obtained.ResultsPart One, CCI model establishmentThe ipsilateral threshold of mechanical hypersensitivity and thermal allodynia dropped from the third day, and declined to the lowest at the tenth day but kept the stable level during 21 days. And the successful rate of CCI kept up to 80% approximately.Part Two, pain relief assessment of YZPAll doses showed significant pain relief on the fifth day(YZP2, YZP3, YZP4, P<0.01; YZP1,p<0.05) and kept being of efficacy during the administration with dose-dependence. The effect of pain-relief of the four doses of YZP was attenuated with the withdrawal, while the efficacy of YZP1 disappeared on the second day,YZP3 on the fourth day, but YZP2 and YZP4 on the seventh day after drug withdrawal (p>0.05). The tendency of positive drugs gab and LTD on CCI-induced mechanical hypersensitivity was almost identical. In comparison with CCI group, the thresholds of mechanical hypersensitivity began to have significance on the fifth day and lasted on the fourteenth day with positive drugs gab and LTD infusion (p<0.01). However, the positive drugs had no pain relief at the fourth day after the withdrawal.During the first week, the effect of different doses of YZP on CCI-induced thermal allodynia was unstable with no significance (p<0.01), but from the tenth day they showed significant (p<0.01) with dose-dependence. With the withdrawal, almost all doses of YZP had no significance in comparison with CCI group except for YZP3 (p<0.05).The results of the thresholds of thermal allodynia showed that gab and LTD group had no significance with CCI group during the first week (p>0.05), but worked from the tenth day to the fourteenth day (p<0.01). After the withdrawal, both positive drug groups kept analgesia for two days. However, it was interesting that YZP with different doses, gab and LTD had no effect on the thresholds of mechanical hypersensitivity and thermal allodynia for the contralateral paws (p>0.05) during the 21 observing days.Part Three, molecular mechanism of YZP for neuropathic painIn comparison with sham group, only the content of LEU between 4 kinds of amino acids increased markedly (p<0.05), which could be reversed by gab administration (p<0.01). However LTD and YZP with different doses had no effect on CCI-induced the concentration of LEU (p>0.05).32 different expressions of miRNAs and 222 different expressions of mRNA were gained through miRNA microarray and Gene microarray analysis respectively. Then, these miRNAs were predicted in programs for their predicted targeting mRNAs, which intersected with 120 different expression mRNAs.15 couples of miRNAs and mRNAs were obtained. And it was interesting that 5 couples of miRNAs and mRNAs were possible to be associated with neuropathic pain through literature searching, including miR-301 a and CASR, miR-301 a and NPTX1, miR-96-3p and NPTX1, miR-411-3p and AKAP5 and miR-543-5p and AKAP5.Conlusion1.CCI model was established successfully in the study. YZP was determined to attenuate CCI-induced mechanical hypersensitivity and thermal allodynia with dose-dependence.2.In comparison with sham group, the concentrations of four kinds of amino acids had no changes in CCI group, except LEU. But both LTD and YZP could not attenuate CCI-induced increased concentrations of amino acid, suggesting that it was possible that YZP may not modulate amino acid in cerebrospinal fluid.3.One of molecular mechanism of YZP for the treatment of neuropathic pain may be corresponding to miRNAs in DGR, which mediated their targeting genes, involving miR-301a and CASR, miR-301a and NPTX1, miR-96-3p and NPTX1, miR-411-3p and AKAP5 and miR-543-5p and AKAP5.