Effect Of Chuanjiao Decoction On Mast Cell Degranulation In Allergic Conjunctivitis And Its Mechanism

Clinically, allergic conjunctivitis(AC) is one of the most common ocular conditions accompanying with allergic rhinitis, asthma. AC is type I hypersensitive, which is the result of ocular exposure to an antigen that causes cross-linkage of membrane-bound immunoglobulin E (IgE), resulting in mast cell(MC) degranulation. MC degranulation causes the release of preformed mediators such as histamine, as well as newly formed MC mediators such as prostaglandins. Currently, antihistamines, MC stabilizers and anti-inflammatory agents including steroids and nonsteroidal drugs are conventionally used for the treatment of AC. However, antihistamines and MC stabilizers cannot suppress the activation of free neutrophils and eosinophils. Long-term use of glucocorticosteroids can cause serious side effects such as glaucoma, cataract, Cushing’s syndrome, osteoporosis and bacterial infections. Anti-inflammatory and steroid medicine often show side effects with long treatment duration. AC has been one of the challenging issues in terms of ophthalmology because of the high incidence of the disease, long duration, easy to break out repeatedly, limited curative effect.Eye, as one of the organs, participates in systemic anaphylaxis with ocular tissues. Animal mAels confirmed that ovalbumin (OVA) delivered systemically hypersens-itivity could also reach ocular mast cells and thereby elicit their participation in the systemic reaction,and appeared ocular symptoms.Moreover, AC is always a comorbid condition of allergic rhinitis and asthma. It was concluded that AC is one symptom of systemically hypersensitivity in eyes, and it should be treated from the whole. Chuanjiao Decoction is the experience of the AC treatment from Professor Gao Jiansheng, the famous ophthalmologist of traditional Chinese medicine (TCM). Under the guidance of philosophical thinking of TCM, according to TCM and clinical practice, professor Gao put forward the idea of TCM pathogenesis of AC is "intertwist of cold with heat",and he summarized that the Chuanjiao Decoction has good curative effect on AC and other type I hypersensitive, such as asthma and allergic rhinitis. In our previous studies, we have proved that the Chuanjiao Decoction, which consisted of herba schizonepetae,radix saposhnikoviae,fructus kochiae, fructus cnidii, rhizoma ligustici chuanxiong,rhizoma anemarrhenae,pericarpium zanthoxyli, was found superior to sodium cromoglicate and olopatadine in reducing pruritus, hyperemia, chemosis,tearing, and lid swelling of patients. In animal model,Chuanjiao Decoction has active effect on control of acute symptom of AC by OVA; reduce the level of IgGl,IgE and IL4in blood serum of mice; and also has positive impact on change of mast cell and reduction of chemotaxis on eosinophile and neutrophile granulocyte, which restrained late reation. We concluded that Chuanjiao Decoction have a role of "stable mast cell and inhbit degranulation". But its detailed mechanism remains unclear. Therefore,aiming the two main objectives in the treatment and prevention of AC, this study was designed to investigate the effect Chuanjiao Decoction on MC and its potential mechanisms. Meanwhile, if this mechanism is confirmed, it would be a new strategy for the treatment of both allergic conjunctivitis and other allergic diseases such as asthma and allergic rhinitis in traditional Chinese medicine,and support the idea of "intertwist of cold with heat"Experimental StudyObjective:To observe the effects of Chuanjiao Decoction(CJD) on mast cell(MC) degranulation in allergic conjunctivitis (AC), and clarify the mechanism.Methods:(1)Totally84Balb/c mice were randomlly divided into the normal group(12mice), the mAel group(12mice), the placebo group(12mice), the0.1%olopatadine eye drops group(12mice), the low,medium,and high dose CJD groups(12mice). After induced allergic conjunctivtis by intraperitoneal injection ovalbumin, mice in the normal group and mAel group were not gastrogavage, mice in the placebo group were given10mL-kg~1normal saline by gastrogavage,once daily for7days. Mice in the0.1%olopatadine eye drops group were given with0.1%olopatadine eye drops twice a day for7days.Mice in the low, medium,and high dose CJD groups were given with CJD10mL-kg~1at the dose of5.25,10.5,21.0g-kg~1by gastrogavage, once daily for7days. Mice were stimulated again after the above treatment, and their eyes’acute clinical effect were observed. The conjunctiva tissue were dyed by toluidine blue staining, and the number of total MC and degranulated MC were counted. Expression of substance P (SP)was detected using immunohistochemisty assay.The concentration of SP, prostaglandin D2(PGD2), histamine (HIS) and tumor necrosis factor-alpha(TNF-a) in mice serum were measured by ELISA. The mRNA expression of interleukin4(IL-4)and TNF-a in mice conjunctiva were detected using RT-PCR.(2) Rat in the low, medium,and high dose CJD groups were given with CJD20mL-kg~1at the dose of6.68,13.36,26.71g-kg~1by gastrogavage, twice daily for3days. Rat in the placebo group were given20mL·kg-1normal saline by gastrogavage, twice daily for3days. Preparation of SD rats serum containing Chuanjiao decoction, and the content of osthole in serum was determinated by high performance liquid chromatography (HPLC). The effects of CJD at different concentration on cell vitality was observed by methyl thiazolyl tetrazolium(MTT) assay.(3) For cell stimulation, cells were primed for4h or overnight with different concentration of DNP-specific IgE. Cells were washed and resuspended in a PIPES-buffered medium for RBL-2H3cells. Then adding25ng/mL antigen (DNP-BSA) in RBL-2H3cells for the following experiments. Degranulation was determined by measuring release of the granule marker, β-hexosaminidase in media, through a colorimetric assay in which release of p-nitrophenol from p-nitrophenyl-N-acetyl-β-D-glucosaminide was measured. Values were expressed as the percentage of intracellular β-hexosaminidase that was released into the medium.According to the β-hexosaminidase in media, determine the optimal concentration of antibody, antigen action time and the function of the medicated serum best time.(4) After induced RBL-2H3cells degranulation by antigen. the number and the ratio of degranulated MC were determinated using toluidine blue staining method; The levels of TNF-α and IL-4in the culture media were determined using commercial ELISA kits. The levels of TNF-α and IL-4mRNA were determined using commercial real time PCR. Immunoblot analysis was also used to detect the phosphorylated forms of AKT and MAP kinases in the whole cell lysates.Result:(1) Compared with the normal group, scores of signs, the number of total MC and degranulated MC were increased in the model and placebo group (P<0.05,P<0.01); the expression of SP, PGD2, HIS and TNF-a in mice serum significantly increased in the mAel and placebo group(P<0.01). the expression of SP, IL-4and TNF-α in mice conjunctiva significantly increased in the model and placebo group(P<0.01). Compared with the model and placebo group, the number of total MC and degranulated MC were decreased in the CJD groups (P<0.01); the expression of SP, PGD2, HIS and TNF-α in mice serum significantly decreased in the CJD groups (P<0.01); the expression of SP, IL-4and TNF-α in mice conjunctiva significantly decreased in the CJD groups(P<0.01). There was no statistical difference among the CJD groups and Olopatadine group (P>0.05). (2) HPLC showed that CJD serum solution retention time is between6.15to6.55min. The blank serum solution in this time period without obvious absorption peak. The effects of CJD on cell activity is relationship with action time and concentration. MTT shows that10%concentration of serum is better, and we chose the10%concentration of serum for experiment.(3) Compared with other concentration of antibody,and antigen action time, the release of β-hexosaminidase in media significantly increased (P<0.01),when cells were primed for with25ng/mL of DNP-specific IgE, and adding25ng/mL antigen (DNP-BSA) in RBL-2H3cells for15min. Cell incubation with serum120min,before adding antigen, the release of β-hexosaminidase in media significantly decreased (P<0.01)(4) Compared with the normal group, the ratio of degranulated MC were increased in the mAel and blank seurm group (P<0.01); the release of β-hexosaminidase in media were significantly increased in the model and blank seurm group (P<0.01); the expression of IL-4and TNF-a in media were significantly increased in the model and blank seurm group (P<0.01); the mRNA levels for IL-4and TNF-a were significantly increased in the model and blank seurm group (P<0.01); the phosphorylated forms of Akt, and MAP kinases in the whole cell lysates were increased in the model and blank seurm group (P<0.01). Compared with the model and blank seurm group, the ratio of degranulated MC were decreased in the CJD groups and the PP2group(P<0.0l); the release of β-hexosaminidase in media were significantly decreased in the CJD groups and the PP2group (P<0.01); the expression of IL-4and TNF-a in media significantly decreased in the CJD groups and the PP2group (P<0.01); and the CJD groups and the PP2group significantly decreased IL-4and TNF-a mRNA levels, and AKT, JNK, ERK, p38phosphorylated protein level in Ag-stimulated RBL-2H3cells. The inhibitory effect of CJD on mast cell degranulation in a concentration dependent manner.Conclusion:(1)The action mechanism of CJD in treating AC might be achieved through stabilized and inhibited degranulation of mast cells.(2) The role of "stable mast cell and inhbit degranulation" might be achieved through decreased AKT, JNK, ERK, p38phosphorylated protein level by Chuanjiao Decoction.