The Anti-inflammatory Effect Of KuAnQuZhen Gelatum And Its Effect On Rat Model Of Eczema

Background:Eczema is a common allergic skin disease. It has the characteristics of severe itching, rash, and tendency of exudation. And eczema is more difficult to treat. Due to the loss of skin and severe itching, the patient’s psychological, dressing, social and entertainment, work study and treatment are affected in different degree, which seriously affect the quality of life. Domestic and foreign studies indicate that eczema patients’ 90 symptom score somatization, interpersonal relationship, horror, depression, anxiety, paranoid factor score has statistical significance, and intense itching and depression and anxiety may have a serious effect on patients with sleep.China’s 2011 guidelines for the diagnosis and treatment of eczema pointed out: eczema is a kind of obvious exudation of inflammatory skin disease caused by a variety of internal and external factors, with obvious itching, easy to relapse. Eczema is clinically divided into acute, subacute and chronic phase. Acute stage performance for miliary papules, herpes, blisters, erosion and exudation lesions based on erythema and edema, from the center outward spread. Subacute stage was swelling and exudation, crusting, scaling, erosion surface. Chronic eczema is a manifestation of rough hypertrophy, moss change with hair and nails change, pigment change, consciously itching and even itching. At present, the topical corticosteroid preparation is still the main drug treatment of eczema, which can effectively alleviate the symptoms. But eczema itself is easy to repeat, long-term use of corticosteroids can cause skin atrophy, telangiectasia, pigmentation, sometimes induced infections. Topical calcineurin inhibitors (such as tacrolimus ointment) is a new drug in the treatment of eczema, similar to the role and effect of glucocorticoid, no similar glucocorticoid side effects, but the price is expensive. The therapeutic efficacy remains to be further clinical data support.TCM has a long history in the external treatment of eczema and curative effect is reliable. Eczema’s record scattered in "boil", "wet sores", "ringworm", "wind" in the ancient books. TCM think the cause of eczema is genetic defect, eat too many fatty, spicy, and is close to the heart, spleen, and liver function disorder. TCM has wide indications, less side effects, low drug resistance, the comprehensive curative effect is prominent. KuAnQuZhen Gelatum consists of Eucalyptus robusta, cnidium fruit, flavescent sophora root, Guangdong redbud bark, patchouli, Tripterygium wilfordii, borneol. Research shows that the Eucalyptus robusta for the treatment of burns, cellulitis, mastitis, eczema, tinea pedis. Eucalyptus robusta of different solvent extracts on Staphylococcus aureus, Escherichia coli, Bacillus subtilis and other bacteria and green wood mold, green mold and other fungi have different degrees of inhibition. Patchouli has obvious inhibitory effects on Candida albicans, Cryptococcus neoformans and other fungi, has certain antioxidant capacity. Matrine for diarrhea, hematochezia, jaundice, urinary, leucorrhea, swelling of the vulva, vulva itch, eczema, sore, itchy skin, scabies, leprosy. Matrine has antifungal activity in vitro. Guangdong redbud bark in the treatment of skin itching, eczemaThe pathogenesis of eczema, inflammatory cytokines play an important role. Thl/Th2 cell differentiation caused by unbalanced cytokine secretion is one of the pathogenesis of eczema, Thl cells secreted IFN-γ and Th2 cells produce IL-4, the examination of IFN-y and IL-4 level are used to reflect the Thl/Th2 cell differentiation. Eczema showed an advantage in Th2 cell response, IL-4 increased, resulting in increased expression of endothelial cell adhesion molecules, chemotaxis leukocyte aggregation to produce inflammation. Leukotrienes are inflammatory mediators play an important role in the pathogenesis of eczema, combined with cysteinyl leukotriene receptor, causing the occurrence of a variety of allergic reactions. The study showed that the levels of LTB4 and LTC4 in the peripheral blood of patients with eczema were increased, and the CysLTRl and CysLTR2 of skin tissue were increased. LTS acts on receptors, white blood cells are chemotaxis, causing skin inflammation, the performance of skin erythema, swelling, exudation, caused by skin lesions. LTS can increase capillary permeability, capillary blood plasma exudation to form edema, showing swelling of the skin, exudation. IL-17 and IL-31 are found in recent years, with eczema related inflammatory factors, the study found that eczema patients and animal models of IL-17 are elevated, IL-17 as an early cytokine induced immune cells to release pro-inflammatory factors. The effect of IL-31 on the receptor, epidermal keratinocytes induced production of inflammatory cytokines. Inflammatory cytokines causes the skin to produce an inflammatory response, causing eczema like skin lesions. And IL-31 is closely related to itching.Researc group for many years engaged in clinical skin, combined with the southern region of the law and characteristics of skin diseases in the humid and hot climate environment, create a prescription "XiaoZzhenZhiYang paste". It consists of Eucalyptus robusta, cnidium fruit, flavescent sophora root, Guangdong redbud bark, patchouli, Tripterygium wilfordii, borneol, for the treatment of eczema, dermatitis, psoriasis, eczema acute, sub acute eczema, chronic eczema, neural dermatitis, atopic dermatitis. On the basis of previous studies, with "XiaoZzhenZhiYang paste" as the foundation, extracting the effective parts, the development of KuAnQuZhen Gelatum in efficacy study has confirmed that KuAnQuZhen Gelatum has bacteriostasis, anti allergic and anti pruritus effect. This study will explore the anti-inflammatory effects of KuAnQuZhen Gelatum and the role of eczema model, focusing on its impact on IFN-γ, IL-4, IL-17, IL-31, LTB4, LTC4, and the role of CysLTR1 and CysLTR2, clear the mechanism of its action.Objective:1. To reseach the anti-inflammatory effect of KuAnQuZhen Gelatum by studying the effect of KuAnQuZhen Gelatum on rat foot swelling model, mouse ear swelling model, and skin capillary permeability increasing model.2. To study the effect of KuAnQuZhen Gelatum on the model of DNCB induced eczema in rats, and to explore its mechanism, to lay the experimental foundation for the further development and utilization of KuAnQuZhen Gelatum.Methos:1 Experimental drug:KuAnQuZhen Gelatum, gelatum substrate, PiYanPing ointment, BingHuangFuLe ointment.2 Experimental animals:120 KM mice,100 SD rats. Male and female half.3 The study of Anti-inflammatory effect of KuAnQuZhen Gelatum3.1 The rats foot swelling experiment:60 rats were randomly divided into model group, gelatum substrate group, PiYanPing ointment group, KuAnQuZhen Gelatum low dose group, KuAnQuZhen Gelatum midle dose group, KuAnQuZhen Gelatum high dose group. In the rat in the right foot dorsal applicator for 7 days. After coated medicine, the right rear foot was subcutaneous injected carrageenan. The volume of the foot before and after injection were measured.3.2 Mouse ear swelling test:60 mice were randomly divided into model group, gelatum substrate group, PiYanPing ointment group, KuAnQuZhen Gelatum low dose group, KuAnQuZhen Gelatum midle dose group, KuAnQuZhen Gelatum high dose group. Applicator on the mouse right ear for 7 day, then in the right ear drops by xylene solution, punch in the left and right profile similar position lay round the ear piece and weight. The difference of left and right ear quality is the degree of swelling. Make pathological section to observe the change of inflammation.3.3 Mice increase of blood capillary permeability of the skin:60 mice were randomly divided into model group, gelatum substrate group, PiYanPing ointment group, KuAnQuZhen Gelatum low dose group, KuAnQuZhen Gelatum midle dose group, KuAnQuZhen Gelatum high dose group. Applicator on the mouse back skin for 7 day, then intravenous injection of saline 0.5% Evans blue. Apply xylene, cut off the skin and put in the acetone water (7:3), and measure the absorbance in the 590nm wavelength range of the spectrophotometer.4 Effect of KuAnQuZhen Gelatum on rat eczema model skin:40 rats were randomly divided into normol group, eczema model group, KuAnQuZhen Gelatum group, positive control group. At the back of the rat skin with DNCB acetone solution sensitized and excited to make the model of eczema. After treatment, the record of skin lesions and score were detected. Take the skin to mak pathological sections.5 Effect of KuAnQuZhen Gelatum on eczema related cytoldnes and CysLTRl and CysLTR2:The serum levels of IFN-, IL-4, IL-17, IL-31, LTB4, LTC4, and were detected. Immunohistochemistry and Westernblot method were used to detect the expression of CysLTR1 and CysLTR2 in skin lesions.6 Statistical analysis:Using x±S for statistical description, using single factor analysis of variance, multiple comparison using LSD method, P<0.05 has statistical significance.Result:1. Compared with the model group, foot swelling of gel matrix group, piyanping ointment group, KuAnQuZhen Gelatum low, middle and high dose group were relieved at 1 h,2 h,3 h and 4 h (P< 0.01). No statistical difference (P>0.05) in low, middle and high dose group foot swelling comparison.2. Compared with the model group, ear swelling showed no significant difference (P>0.05) in the gel matrix group and BingHuangFule ointment group. KuAnQuZhen Gelatum low, medium and high dose group ear swelling reduced (P<0.05). No statistical difference (P>0.05) in low, middle and high dose group ear swelling comparison. Inflammatory cell infiltration and intercellular edema in KuAnQuZhen Gelatum group were decreased.3. Compared with the model group, there was no significant difference in the absorbance of the gel matrix group, Binghuangfule ointment group (P>0.05). KuAnQuZhen Gelatum low, medium and high dose group were decreased (P＜0.05). No statistical difference (P>0.05) in low, middle and high dose group comparison.4. Compared with eczema model group, the skin lesion score in group A was decreased at sixth days, eighth days and ninth days (P＜0.05). It showed erythema, relief, papules, scratches/stripping off reduced. In the positive control group, the skin lesion score was decreased (P＜0.05) in sixth days.5. The pathological sections showed the eczema model group showed hyperkeratosis, parakeratosis, acanthosis, hypertrophy of granular layer. The pathological changes of KuAnQuZhen Gelatum group and positive control group decreased.6. There was no significant difference in the level of IFN-yin serum of each group (P>0.05). Compared with the normal group, the level of IL-4 in eczema model group was increased (P＜0.05). Compared with the eczema model group, the IL-4 levels of of KuAnQuZhen Gelatum group and positive control group drop (P＜0.05).7. Compared with the normal group, the levels of IL-17, IL-31, LTB4 and LTC4 in eczema model group were increased (P＜0.05). Compared with eczema model group, the levels of IL-17, IL-31 and LTC4 in KuAnQuZhen Gelatum group were decreased (P＜0.05), but there was no significant difference in LTB4 level (P>0.05). Positive control group of IL-17, IL-31, LTB4, LTC4 levels decreased (P＜0.05).8. Results of immunohistochemistry:Compared with the normal group, eczema model group CysLTRl and CysLTR2 were increased, which mainly expressed in the skin of the epidermis and dermis. Compared with the eczema model group, CysLTRl and CysLTR2 expression were down regulated in the KuAnQuZhen Gelatum group and the positive control group.9. Western Blot results:Compared with the normal group, the model group showed high expression of CysLTRl (P<0.05) and CysLTR2 (P＜0.01). Compared with eczema model group, the expression of CysLTRl in KuAnQuZhen Gelatum group was decreased (P＜0.05), and the expression of CysLTR2 was decreased (P＜0.01).Compared with the positive control group, the expression of CysLTR1 and CysLTR2 in KuAnQuZhen Gelatum group had no statistical difference (P>0.05).Conclusion:1. KuAnQuZhen Gelatum has anti-inflammatory effect.2. KuAnQuZhen Gelatum improves the skin lesions in rats with eczema, which is related to the down-regulation of IL-4, IL-17, IL-31, LTC4 levels in serum and down regulate the expression of CysLTRl and CysLTR2 in the skin.