| Objective: Pruritus is a persistent symptom commonly found in many diseases,which seriously impairs patients’ physical and mental health as well as the quality of life,and its treatment and care have become a clinical problem of focus.According to the "Compendium of Materia Medica" and other classics,the root bark from Broussonetia papyrifera is widely used to treat dermatoses such as dermatitis and ringworm.Modern studies have also confirmed the anti-inflammatory and antimicrobial effects of the root bark from Broussonetia papyrifera,but its antipruritic effects and related mechanisms have not been clarified.This study aimed to explore the antipruritic effect of the root bark from Broussonetia papyrifera and its related mechanism to provide a theoretical basis for the clinical treatment and nursing of pruritic patients.Methods: 1.The ethanol extraction and process optimization of the root bark from Broussonetia papyrifera The root bark from Broussonetia papyrifera was extracted by the ethanol reflux extraction method.To optimize the extraction conditions,single-factor experiments and response surface methodology with total flavonoids and polyphenols as evaluation indicators were used.2.Screening antipruritic effective extract fraction of ethanolic extract from Broussonetia papyrifera root bark The ethanolic extract of the root bark from Broussonetia papyrifera was extracted with ethyl acetate and water-saturated n-butanol sequentially.Mice were randomly divided into the normal group,model group,ethanol extract group,ethyl acetate extract group,n-butanol extract group,and water phase extract group.Different extracts were prepared into hydrogels and applied locally on mouse skin for 7 consecutive days.After the last administration,except for the normal group,the other mice were injected subcutaneously 4-aminopyridine(4-AP)to induce acute itch,and the change of scratching behavior and the total number of scratches within 30 min were recorded and counted to compare the antipruritic effect of different extraction sites of root bark ethanol extract.The high-performance liquid chromatography and ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry were used to analyze the substance components of the antipruritic effective extract fraction. 3.Exploration of the antipruritic effect and mechanism of the effective extract fraction Mice were divided into normal group,model group,and groups with different doses of the effective extract fraction,and were topically applied with hydrogel for 7 days.After the last administration,except for the normal group,the other mice were injected subcutaneously with 4-AP and chloroquine(CQ)to induce histamine-dependent pruritus and non-histamine-dependent pruritus,respectively.And the change in scratching behavior and the total number of scratches within 30 min were recorded and counted.Mice were divided into the normal group,model group,and high or low dose of effective extract fraction group.Except for the normal group,the other mice were applied locally for 7 consecutive days to establish a mouse chronic itch model by applying an acetone-ether mixture and distilled water(AEW),and continuously applied the hydrogel for 4 days from the 4th day of modeling.The total number of scratches in mice within 1 h on days 1,3,5,and 7 was recorded and counted.The changes in the appearance of the dorsal skin in mice were photographed and the histopathological analysis of the skin was performed by hematoxylin-eosin staining.The contents of Interleukin-4(IL-4),IL-6,and Tumor necrosis factor-α(TNF-α)in the serum and skin of mice were detected by enzyme-linked immunoassay.Western blot was used to analyze the expression of nuclear factor κB(NF-κB)subsets p65,phosphorylated p65(p-p65),and transient receptor potential vanilloid 1(TRPV1).On this basis,the AEW-induced chronic itch mouse model was established,which was divided into the normal group,model group,TRPV1 inhibitor group and the effective extract fraction combined with TRPV1 inhibitor group,and the number of scratches in each group was recorded and the expression of TRPV1 was detected by Western blot.Results: 1.The optimal conditions for ethanol reflux extraction of the root bark from Broussonetia papyrifera The optimum conditions for ethanol reflux extraction of the root bark from Broussonetia papyrifera were: extraction temperature 75 °C,extraction time 117 min,solid-liquid ratio 1:16(g/m L),and ethanol concentration 70%.Under these conditions,the actual extraction amounts of total flavonoids and polyphenols were(23.93±0.30)mg/g and(14.69±0.56)mg/g,respectively.2.Effective extract fraction for itch relief of ethanol extract from Broussonetia papyrifera root bark The ethanol extract of the Broussonetia papyrifera root bark was further extracted,and ethyl acetate extract,n-butanol extract as well as water phase extract were obtained.The total number of 30 minutes of scratching in the model group was significantly higher than that in the normal group(P<0.05).Compared with the model group,the total number of scratching times in the ethanol extract group and n-butanol extract group was significantly reduced from the 10 th minute,and the total number of scratches in the 30 minutes was significantly decreased(P<0.05).However,there was no significant difference in the number of scratches in the ethyl acetate extract group and the water phase extract group compared with the model group(P>0.05).There were 10 major peaks in the fingerprint of n-butanol extract,of which peak 7 has the largest reserved area,accounting for 36.01%.UPLC-QTOF-MS/MS analysis showed that the substance of peak 7 was chlorogenic acid.3.The antipruritic effect and mechanism of the effective extract fraction In the two acute itch models induced by 4-AP and CQ,the high-frequency scratching behavior of mice in the model group was more than that in the normal group,which began to decrease after 25 minutes(P<0.05),and the total number of scratches in 30 minutes was significantly higher than that in the normal group(P<0.05).After treatment with the medium or high doses of the n-butanol extract,the scratching behavior of mice was significantly decreased at the 10 th or 15 th minutes,and the total number of scratches at 30 minutes was significantly lower than that of the model group(P<0.05).In AEW-induced chronic itch,the number of scratches in the model group increased day by day within 7 days(P<0.05).The skin of mice in the model group was dry and peeling with the significantly increased epidermal thickness(P<0.05).Besides,compared with the normal group,the levels of IL-4,IL-6,and TNF-α in the serum and skin of mice in the model group were significantly increased,and the expression of p-p65 and TRPV1 in the skin was significantly increased(P<0.05).Compared with the model group,after treatment with different doses of the n-butanol extract,the number of scratches in mice was significantly reduced from day 5(P<0.05),the degree of skin dryness was decreased,and the thickness of the epidermis was significantly reduced(P<0.05).Meanwhile,the contents of IL-4,IL-6,and TNF-α in the serum and skin of mice were also decreased(P<0.05),and the expression of p-p65 and TRPV1 in the skin was significantly reduced(P<0.05).Compared with the TRPV1 inhibitor group,mice in the TRPV1 inhibitor with the n-butanol extract group had fewer scratches within 1 h at the end of the experiment(P<0.05),but there was no difference in TRPV1 expression in the skin between the two groups(P>0.05).Conclusion: 1.The n-Butanol extract is the antipruritic effective fraction of the root bark from Broussonetia papyrifera,and its most abundant substance component is chlorogenic acid.2.The n-butanol extract of the root bark from Broussonetia papyrifera had inhibitory effects on histamine-dependent or non-dependent acute pruritus induced by 4-AP or CQ,and also alleviated chronic itch caused by AEW,reducing skin dryness and epidermal thickening in chronic itch mice,which might be related to the inhibition of NF-κB pathway,regulation of inflammatory response,and reduction of TRPV1 expression and itch signaling. |
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