Gentiopicroside Reduces Pain,Pruritus,and Corticosteroid Addictive Dermatitis

Abstract

Corticosteroid addictive dermatitis (CAD) has rapidly emerged as a health problem which is difficult to cure. The dermatitis control is general with the tacrolimus, pimecrolimus, and antihistamines, and these synthetic drugs are likely to have some side effects, and how to use the nonirritating natural product to reduce the CAD has been rarely reported. Strong evidence indicates that gentiopicroside (GPS) has been reported to have anti-inflammation and anticancer properties. In the present study, we invented a device to collect GPS to study the effect of GPS on pain, pruritus, and CAD repair in model animals. Our results showed that the data on antipain and antipruritus treated with GPS were better than those of control group, and the inflammation of rabbit skin upon 2,4-dinitrochlorobenzene exposure reduced by GPS. In conclusion, GPS could be a factor for antipain, antipruritus, and CAD repair; hence, these findings suggest that it can act as a protective factor for CAD.

Dermatitis, a common inflammatory skin disease, is caused by a variety of internal and external factors in the epidermis and superficial layer of the dermis. It is characterized by severe itching, skin lesion multiform, symmetrical distribution, exudative tendency, chronic course of disease, and easy to relapse. Corticosteroid addictive dermatitis (CAD), caused by repeated inappropriate external hormone, has rapidly emerged as a health problem which is difficult to cure. The dermatitis control in clinic is general with the tacrolimus, pimecrolimus, and antihistamines. However, these synthetic drugs are likely to have some side effects, and how to use the nonirritating natural product to reduce the CAD has been rarely reported. Strong evidence indicates that gentiopicroside (GPS) from Gentiana scabra Bunge has anti-inflammation and anticancer properties. ^1,2 In the present study, we invented a device to collect GPS to study the effect of GPS on pain, pruritus, and CAD repair in model animals. Our results in mice have shown that the data on antipain and antipruritus treated with GPS were better than those of control group, and the inflammation of rabbit skin upon 2,4-dinitrochlorobenzene exposure reduced by GPS. Herein, GPS provided significant improvement in symptom severity.

In this study, we combined the expansion bed extraction technology (Figure 1(a)) and pulsed strengthening technology (Figure 1(b)) to solve the problems such as less effective ingredients and more impurities in the extraction of traditional plant compounds (GPS from G. scabra Bunge, Figure 1(c)). The whole production process ensures safety, environmental protection, and low energy consumption. The extracted liquid was stable and easy to change. Compared with the traditional method, the purification efficiency improved obviously. The results of fingerprint by HPLC were performed 3 times well repeated in Figure 1(d). To clearly identify the pain threshold, anti-irritation, pruritus frequency, and histamine tolerance of GPS on animals, we initially treated mice with GPS at different tests. As shown in Figure 2(a-g), GPS reduced anti-irritation, pruritus frequency, and histamine tolerance; meanwhile, GPS improved pain threshold like Votalin. Our results also provided further evidence on tissue architecture. Improvement with GPS was found using the hematoxylin and eosin (HE) stain, which was involved in anti-inflammation properties, since lymphocyte number in mice was closely related to the regulation of dimethylbenzene and hydrocortisone. Although the GPS was identified as a drug candidate, no clinical case demonstrated that GPS acted in a CAD manner. In addition, the treatment of GPS on dermatitis has been identified by volunteers in clinical cases, for example, CAD, dermatitis, and chronic eczema. Although GPS, like dexamethasone, was a useful drug to reduce inflammation on rabbit in this study (Figure 3), GPS might also serve as a therapeutic option underlying the common dermatitis that remains largely unknown because of complex inheritance.

Figure 1

Gentiopicroside extract transdermal manufacture and the test system. (a) Expansion bed extraction device. (b) Fixed bed extraction device. 1, Tank; 2, sintering plate distributor; 3, first pump; 4, second pump; 5, current diverter; 6, raw material particles. (c) Gentiopicroside and Gentiana scabra Bunge. (d) Fingerprint and the content of demarcated objects by HPLC, 3 repeated experiments.

Figure 2

Identification of animal test and immunohistochemistry: (a) Pain threshold results, (b) anti-irritating results, (c) pruritus frequency results, (d) histamine tolerance results, (e) inflammation and immunohistochemistry results of lymphocyte number, (f) the analysis on thickness of ears and (g) the quantification of lymphocyte number.

Figure 3

Gentiopicroside reduced 2,4-dinitrochlobenzene-induced inflammation and treatment in rabbit skin.

Gentiana scabra Bunge contains gentiopicrin, swertiamarin, sweroside, trifloroside, amarogentin, gentiopicroside tetraacetate, amaroswerin, gentioflavine, gentianine, gentianidine, gentianol, gentianose, β-sitosterol, and vitamins, and it is considered as a rich source of antioxidant. Gentiopicroside, the main component in G. scabra Bunge extract, has strong hydrophilic property, and the lipophilic activity is relatively poor. As a natural iridoid glycoside, GPS had also been widely used as a therapeutic option and had the effect on gallbladder, anti-inflammation, stomach invigoration, and depressurization. ^3,4 Gentiopicroside had been reported to show a potent protective effect on TNF-α and IL-1β induced inflammation response. ^5,6 The numerous enzyme genes in the glycolysis pathway were involved in GPS-mediated process. ⁷ Furthermore, GPS had been reported for regulating P2X7R-mediated inflammatory response via LKB1 and AMPK. ⁸

Our results demonstrated that GPS inhibited pain and pruritus in an anti-inflammatory manner in vivo, and the effect of GPS on dermatitis had no report in the previous studies. Moreover, the GPS exhibited repair function during the skin damage. This association expanded the involvement of GPS in human dermatitis by underlying a role in nonirritating natural product, since AbbVie (Upadacitinib) and Pfizer (PF-04965842) released a positive result on atopic dermatitis in 2018. In conclusion, GPS can be a factor for antipain, antipruritus, and anti-CAD repair; hence, these findings suggest that it can act as a protective factor for CAD.

Experimental

ICR Mice, Rabbits, and Guinea Pigs

For animal experiments, mice, rabbits, and guinea pigs were used, and all procedures were performed according to the institutional guidelines for the care and use of laboratory animals of Huaihe Hospital of Henan University. Preparation of animals for test was done as described previously. ^9,10

Skin Irritation Test

A total of 12 healthy guinea pigs were taken for the test. The guinea pigs with no skin damage were provided for this experiment as a control. Before the experiment being performed, the fur was removed and the area was about 3 cm × 3 cm. A total of 0.5 mL specimen was coated on side of the depilation area, and the coating area was about 2.5 cm × 2.5 cm. The medicine was fed once a day for 14 days. Starting from the second day, the hair was cut and the remaining samples were removed. After 1 hour, the results of local skin reaction were observed.

Scratch Test

The back hair of mice had been removed and the drug (0.2 mL) was smeared onto them. Five days after the last administration, dextran 0.95 mg/kg was injected into the tail vein. Scratching the head of the front paw, scratching the trunk of the hind paw, and biting all parts of the body were used as itching indications. The frequency of itching in mice within 30 minutes was recorded, and the differences between the groups were compared.

Histamine Tolerance Test

Right hind feet of guinea pigs were depilatory, and the experimental groups were smeared 4 times a day and 2 times a day. On the third day, the skin of the hind right foot was rubbed gently with fine sand paper, but without bleeding. Then, the drug was smeared onto the wound. After 40 minutes, the drug was gently removed. Histamine was applied to each guinea pig’s hind foot.

Delayed Allergic Reaction

The back hair of mouse has been removed, and dinitrochlorobenzene (DNCB) acetone was applied on that area. Five days later, the right ear was smeared with 20 μL of 0.1% DNCB solution for 5 times (1 time/3 days). From the day of sensitization, the mice were given topical medication, 0.2 mL each time for 14 days. They were smeared on the sensitized area of the back and the inner and outer surfaces of the right ear, respectively. After stimulation for 48 hours, the inflammation of the back skin was observed. After the last dose of 12 hours, the mice were treated as follows: (1) 3 different parts of the skin on the back of the mice were selected. The thickness of the skin was measured with 8 mm metal perforator immediately after the subcutaneous adipose tissue was removed. (2) The skin samples of back were fixed with formaldehyde, paraffin embedded, sectioned, HE stained, and the lymphocytes were counted.

Auricle Swelling Test

Preparation of animals for test was done as described previously. ^9,10 Briefly, 90 healthy guinea pigs, weighing 18 to 22 g, were divided randomly into 9 groups, with distilled water as negative control group. The animal dorsal fur was removed with 1% sodium sulfide, and the area of depilatory skin was 2 cm × 2 cm for 2 days. In the experiment, 0.2 mL of drugs were smeared onto them. Five days after the last administration, dextran 0.95 mg/kg was injected into the tail vein for every 30 minutes in mice. Scratching the head, scratching the trunk, and biting the body were used as itching indications. The frequency of itching within 30 minutes was recorded and the difference between the groups was compared. K12, lauryl sodium sulfate; MTI, methylisothiazolinone; AP, Paeonia lactiflora Root Extract/Paeonia suffruticosa Root Extract; SG, Sophora angustifolia Root Extract/Glycyrrhiza inflata Root Extract/Scutellaria baicalensis Root Extract; Ba, Basam; Fu, Fucosorb; GPS, gentiopicroside.

Hematoxylin and Eosin Strain

In this study, HE was used to check changes in pathological structure. Briefly, the brains were immersed in 4% paraformaldehyde for 6 hours and transferred to 70% ethanol. Individual samples were placed in processing cassettes, dehydrated through a serial alcohol gradient, and embedded in paraffin wax blocks. Before staining, the tissue sections were dewaxed in xylene and rehydrated through decreasing concentrations of ethanol. After staining, samples were dehydrated through increasing concentrations of ethanol and xylene.

Statistical Analysis

The results are shown as the mean ± SD and the significant differences were determined using the two-way analysis of variance. Differences were considered statistically significant if *P < 0.05, **P < 0.01, or ***P < 0.001.