Exploring The Anti-Pruritic And Analgesic Effect Of Sophocarpine In A Co-Morbidity Of Pain And Itch Mice Model

Aim:Chronic pain and itch are different to cure,which affects the quality of life and the physical and mental health of the patients.One in five adults is estimated to suffer from pain,and the prevalence of the chronic itch over 22%.Nowadays,it is hard to cure pain and itch,the treatment process is not only harmful to the body,and even causes serious psychological damage.On the other hand,the patient often suffer form both pain and itch in clinic(co-morbidity pain and itch).The pathophysiological characteristic of comorbidity pain and itch is complex and symptoms involving multiple disciplines,which increase the difficulties of research,even reduces the cure rate in clinic.Sophocarpine(SC)was found from the Radix Sophorae Flavescentis as neuroactive ingredient via CIMC by our research group.In this topic,ICD and ACD mice models were established,after comparing the behavior difference and inflammation intensity the co-morbidity pain and itch model was selected.Next,the co-morbidity pain and itch model was used to investigate the analgesic and anti-pruritic effect of SC and its mechanism.Methods:Experiment part 1: establishment of co-morbidity pain and itch model.SADBE was used to establish the ICD and ACD mice model.Spontaneous pain-like wiping,and itch-like scratching behaviors were recorded one day before and 24 hrs after each challenge.Thermal and Von-Frey type mechanical stimuli were applied to the cheek.Cutaneous inflammation was assessed by ultrasound imaging,measurement of skin thickness,PASI score and histology.Changes in RT-qPCR,immunohistochemistry and western blot for IL-1β,TNF-α,CXCL10 and CXCR3 were evaluated.Experiment part 2: Investigate the analgesic and anti-pruritic effect of SC and its mechanism.Eighty-four male C57/BL6 mice were divided into control,DEM,CPZ,SC 60 mg/kg,30 mg/kg and 10 mg/kg group.SADBE was then used to establish the co-morbidity pain and itch model,on day 10,the corresponding drugs were given by gavage,60 minutes after administration,the mice behavior was recorded for 2 hours.After recording,cutaneous inflammation was assessed by measurement of skin thickness,PASI-Score.The RT-qPCR,western-blot was used to analyze the expression of IL-1β,TNF-α,CXCR3 and CXCL10 on skin,and TRPV1,TRPA1 on TG.Next step,sixty male C57/BL6 mice were divided into control,AITC,AITC+SC,CAP,CAP+SC group.AITC+SC,CAP+SC group was administrated with 10mg/kg SC by gavage,control,ATIC and CAP group was administrated with NS.60 minutes later,AITC and AITC+SC group was intradermal injected with 10μL 1% AITC,CAP and CAP+SC group was intradermal injected with 10μL 1mg/kg CAP,control group was intradermal injected with 10μL 7% Tween-80,then the mice behavior was recorded for 30 minutes.Results:Experiment part 1: Establishment of co-morbidity pain and itch model.Result shown:(1)In comparison with the ICD group,ACD mice had significantly more wipes after each challenge and more scratching bouts after the 2nd and 3rd challenge.There were no gender differences.(2)ACD mice exhibited consistent allodynia and hyperalgesia after each challenge.ACD mice on each day of testing,had significantly greater discomfort scores to 38 or 52 °C stimuli than prior to treatment or than ICD mice.(3)The cheek appeared edematous in both ICD and ACD groups,but more so in ACD,as revealed by skin-fold thickness,ultrasound,and histology.After the third stimulation of ACD group,the stratum corneum increased by 133%.The Hypodermis was 73% in the ICD group.(4)H&E staining showed that both ICD and ACD mice had cutaneous keratinization and increased lymphocyte number,and ACD was more serious than that of ICD mice.The number of inflammatory cell infiltration in ACD group was significantly higher than in ICD group.(5)The expression of IL-1β,TNF-α,CXCR3 and CXCL10 was greater for ACD than ICD.Therefore,ACD which caused stronger pain and itch behavior was selected as co-morbidity pain and itch model for further co-morbidity pain and itching drugs discovery.Experiment part 2: Investigate the analgesic and anti-pruritic effect of SC and its mechanism.Result shown:(1)In comparison with the control group,the mean number of scratching bouts and wipes were significantly higher in model group;SC in different dose could decrease both scratching bouts and wipes.(2)In comparison with the control group,the erythema,scaling and thickening and PASI score was significantly higher in model group;SC in different dose could decrease the PASI score and the skin thickening.(3)In comparison with the control group,the mRNA and protein expression of IL-1β,TNF-α,CXCR3 and CXCL10 in the cheek skin was higher in model group;SC in different dose could down-regulate the mRNA and protein expression of IL-1β,TNF-α but not CXCR3 and CXCL10 in the cheek skin.(4)In comparison with the control group,the mRNA and protein expression of TRPV1 and TRPA1 in the TG was up-regulated in model group;SC could down-regulate the mRNA and protein expression of RPV1 and TRPA1 in the TG.(5)In comparison with the control group,the mean number of wipes were significantly higher in AITC group;SC could decrease both scratching bouts and wipes.In comparison with the control group,the mean number of wipes were significantly higher in AITC group;SC could decrease the wipes.Conclusions:1)ACD mice model had a greater increase in spontaneous itch and pain behavior,hyperalgesia reaction and inflammatory than ICD mice model,the difference may be due to the differential expression of IL-1β,TNF-α,CXCR3 and CXCL10.ACD mice model is more suitable to be used as a co-morbidity of pain and itch model for investigating medicines in the future.This study provides an appropriate animal model for discovering analgesics and anti-pruritic medicines,2)SC had a strong analgesic and anti-pruritic effect in co-morbidity pain and itch mice model,the main mechanism may be due to improving inflammation and inhibiting the expression of TRPV1 and TRPA1 ion channels.It indicates that SC can regulate multiple targets to produce strong analgesic and antipruritic effects.Therefore,this study provides a scientific basis for clarifying the efficacy and mechanism of SC in treatment of co-morbidity pain and itch.