The Relationship Between Oxymatrine Inhibits Neuropathic Pain And HVDCCs Helper Subunits

Purpose:Present study is aimed to observe analgesis of oxymatrine?OMT?against neuropathic pain and further to explore relativity of the mechanism under this OMT analgesis to the auxiliary subunits of high-voltage dependent calcium channels?HVDCCs?.Material and method:1.Through carrying partial sciatic nerve ligation?PSNL?on mice,the neuropathic pain model was established.2.By using von-Frey filament stimulation to detect ED₅₀ of mechanical withdrawing threshold and Catwalk analyzing system to compare Sciatic Functional Index?SFI?among sham operated,PSNL,OMT-administrated mice,analgesia of OMT against neuropathic pain was confirmed on PSNL mice.3.Through real-time PCR method,the influences of OMT on the m RNA expressions of auxiliary subunit ?2?1^?2?4??1^?4??1^?4 were analyzed in the brain,spinal cord and dorsal root ganglia?DRG?tissues from neuropathic pain-modeled PSNL mice.4.Using Western-blotting method,the effect of OMT on auxiliary subunit ?2?1 protein expression was detected in brain,spinal cord and DRG tissues from neuropathic pain PSNL mice.5.With Fluo-3 AM fluorescent probing,the intracellular calcium ion concentration was detected in primary cultured DRG neurons from sham operated,PSNL,OMT,GBP and OMT+GBP-administrated mice,and the effects of OMT and GBP on the intracellular calcium ion concentration were analyzed.6.Using Catwalk analysis system,the changes of SFI,Regularity Index,Swing?s?,Stride Length?cm?,Body Speed?cm/s?,Max Contact Area?cm²?,Print Length?cm?,Print Width?cm?,Print Area?cm²?,Stands?s?,Single Stance?s?,Mean Intensity and Max Contact Max Intensity were analyzed in the sham operated,PSNL,OMT,Gabapentine?GBP?and OMT+GBP-administrated mice,then the effects of OMT and GBP on these Cat Walk parameters were analyzed.Results:1.OMT suppressed neuropathic pain in PSNL mice.Comparing to the sham operated group,the mechanical paw withdraw threshold ED₅₀ was dramatically decreased in the PSNL group mice?P<0.05?;Comparing to the PSNL group,the mechanical paw withdraw threshold ED₅₀?P<0.05?was significantly increased and SFI was remarkably improved?P<0.01?with OMT administration.2.OMT influenced m RNA expressions level of multiple HVDCCs auxiliary subunits in neuropathic pain-modeled PSNL mice.Comparing to the sham operated group,in the PSNL group,the mRNA expression level of HVDCCs auxiliary subunit ?2?1 was remarkably increased in the brain,spinal cord and DRGs?P<0.01?;The m RNA level of ?2?2 subunit was dramatically increased?P<0.05?in the brain and spinal cord but decreased?P<0.05?in DRGs;The m RNA level of ?2?3 subunit was increased?P<0.05?in the brain and spinal cord but decreased?P<0.01?in DRGs;The m RNA level of ?2?4 subunit was decreased in brain?P<0.05?.Comparing to the PSNL group,in the OMT-administrated group,the m RNA expression level of ?2?1 subunit was dramatically decreased in the brain,spinal cord and DRGs?P<0.05?;The m RNA level of ?2?2 subunit was decrease in the brain and spinal cord?P<0.05?;The m RNA level of ?2?3 subunit was decreased in the brain and spinal cord?P<0.01?;The m RNA level of ?2?4 subunit was increased in the brain?P<0.05?.But none of auxiliary subunits ?1-4 and ?1-4 was affected by either PSNL or OMT administration.3.OMT influenced the protein expression level of HVDCCs auxiliary subunit ?2?1 in neuropathic pain-modeled PSNL mice.Comparing to the sham operated group,in the PSNL group,the protein level of ?2?1 was increased in the spinal cord and DRGs?P<0.01?;Comparing to the PSNL group,in the OMT-administrated group,the protein level of ?2?1 was decreased in the spinal cord and DRGs?P<0.01?.Whilest the protein level of ?2?1 didn't change in the brain either in the PSNL or OMT-administrated group.4.Oxymatrine and gabapentine affected intracellular calcium ion concentration in cultured DRG neurons form PSNL mice.Comparing to the sham operated group,the intracellular calcium ion concentration increased significantly?P<0.01?in the cultured DRG neurons from PSNL mice.Whilest OMT,GBP and OMT+GBP administration remarkably suppressed this elevation?P<0.01?.5.OMT and calcium channel regulator GBP suppressed neuropathic pain.Comparing to the sham operated group,in the PSNL group,the sciatie functional index was dramatically decreased?P<0.01?;And to the ligated left side hindpaw in the PSNL group,the parameters to estimate footprints,such as Max Contact Area?cm²?,Print Length?cm?,Print Width?cm?and Print Area?cm²?,and parameters to estimate single paw supporting and pressure,such as Stands?s?,Single Stance?s?,Mean Intensity,Max Contact Max Intensity were remarkably decreased?P<0.01?.Comparing to the PSNL group,to the ligated left side hindpaw in the OMT,GBP and OMT+GBP-administrated group,the Sciatic Functional Index was significantly increased?P<0.01?,and the parameters for estimating footprints?P<0.01?and for single paw supporting and pressure?P<0.01?were remarkably increased.Whilest to the non-ligated right side hindpaw,these parameters didn't show any significant changes in all PSNL,OMT,GBP and OMT+GBP groups.Similarly,none of the parameters to estimate body coordination and balance,such as Regularity Index,Swing?s?,Stride Length?cm?and Body Speed?cm/s?,were affected in any treated group.Conclusion:1.Oxymatrine can improve hyperalgesia and the SFI in naturally traveled neuropathic pain modeled PSNL mice.2.Oxymatrine can dramatically change the m RNA expression levels of HVDCCs auxiliary sunbunits ?2? but not ??? in brain,spinal cord and DRGs from neuropathic pain modeled PSNL mice.Meanwhile,oxymatrine can suppress the PSNL-induced elevation of HVDCCs auxiliary subunit ?2? protein expression in spinal cord and DRGs from PSNL mice.3.Both oxymatrine and calcium channel regulator gabapentine can restrain the elevation of intracellular Ca2+ ion concentration in cultured DRG neurons from PSNL mice,and combination of oxymatrine and gabapentine doesn't show synergistic or complementary effects.4.Both oxymatrine and calcium channel regulator gabapentine can improve same parameters of Catwalk,and combination of oxymatrine and gabapentine treatment doesn't show synergistic or complementary effects.5.Oxymatrine may supress neuropathic pain through regulating HVDCCs auxiliary subunit ?2?.