| OBJECTIVE:The crude herb moxibustion has been widely applied in treating allergic rhinitis in clinical practice which produces the exact effect. Its effectiveness and mechanism has become a research hotspot in recent years, and some achievements has been made. According to the metrology and Meta-analysis results of the application of crude herb moxibustion in treating allergic rhinitis, it is found that previous studies have confirmed its clinical efficacy though, literature of high quality are rare. Previous mechanism researches have touched upon the imbalance relationship in the Th1/Th2of Th cell subsets in AR pathogenesis though, they are not thorough and lack research related to two other subsets of Th cell, namely Treg cells and Th17, which are hotspots in recent immune researches. Currently, the acupoints application drugs for clinical use are tranditional Chinese medicine powder. There exist problem that powder particles in various sizes are difficult to mix evenly, undermining the sticking effect. Therefore, this study proceeds in two aspects. On one hand, it deploys the Chinese herbal submicron powder to destroy the cell wall, which is high bioavailability. The submicron powder used can be fully mixed, conducive to its absorption. The points of dazhui(DU14), feishu(BL13), shenshu(BL23) are selected to treat allergic rhinitis rats and the curative effect is observed. On the other hand, the study explores the possibility mechanism of submicron treatment of AR. Especially on the basis of previous studies, it approaches the therapy of the submicron powder acupoints application on the possible impact of AR rats with Th1, Th2, the subsets Treg and Th17cells in depth, in addition to the interaction between the cell subsets.METHODS:Experimental animails and grouping:A total of56healthly SD rats aged of2months,28males,28females, were fed with six per cage. They were fractionated into two groups according to the random number table principles:10in control group (5male,5female);46in model group (23male,23female). After being adaptive fed for3days,46rats in made module are of intraperitoneal injection with suspension of ovalbumin10mg+Al (OH)3powder30mg+normal saline1ml every other day1/d and a total of7times, as a basis for sensitization. In the15days, we start to strengthen immunity with5%of OVA intranasally50ul/(side, d) and1%of OVA aerosol inhalation5min on consecutive7days. Three weeks later, among46rats in the model group,6died in the process of modeling (2male,4female), Which is considered to be caused by mistakenly intraperitoneal injection into the colon.30minutes after the last excitation, the remaining40rats were observed behavior of scratching nose, sneezing, rhinorrhea in an hour, and were used the superposition method of scoring for behavioral score. All rats’scores were more than five points, showing the model group was successful in all. Successful modeling of the AR model rats were randomly divided into4groups according to the principle of the random number table:10in model group (6male,4female);10in FP (Fluticasone propionate, FP) group (5male,5female);10in group of crude herb moxibustion (5male,5female);10in group of ultrafine herb moxibustion (5male and5female). Five groups of50rats mentioned were examined in the next experiments.Location of Acupoints:The location of rats’ points of dazhui(DU14), feishu(BL13), shenshu(BL23) is in accordance with The Acupoints Positioning of Commonly Used Experimental Animals. The point of dazhui is located between the7th cervical vertebra and the1st thoracic vertebra on the middle of the back. Feishu is below the3rd thoracic vertebra both sides of the intercostals space. Shenshu is located on both sides below the2nd lumbar vertebra.Ordinary powder and submicron powder preparation:The selection of both crude herb and ultrafine herb powder was in line with the specification of Chinese herbal decoction pieces stated in The People’s Republic of China Pharmacopoeia (2005edition). Chinese herbs were produced by Guangzhou Zhixin Chinese Herbal Pieces Co., Ltd. and the lot number was080710.①Crude herb powder:White mustard seed powder, corydalis tuber powder, asarum powder and raw euphorbia powder were configured in the2:2:1:1proportion.②Ultrafine herb powder:The ultrafine herb was more than300orders. That was used of micronized machine to crush the crude herbs powder in proportional allocation by the Second Traditional Chinese Medical Hospital of Guangzhou. The vibrating motor power was1.5FW in15minutes of processing. As a result, the powder was smashed into the size of0.1～75μm and particle size distribution center of D50≈10～15μm.Experimental measures:①Normal control group:no measure was taken;②Model group:saline was given intranasally one time per day for10days;③The fluticasone propionate nasal spray group:the fluticasone propionate aerosol was used to consperge nose1times per day, each50μg/side10days;④The group of crude herb moxibustion(group A):First step was the skin preparation, prorata configured ordinary powder and fresh ginger juice were mixed about0.5cm x0.5cm size of the drugs block. Furthermore, to fix the points with the3cm3cm disposable paper tape, then disposable ventilating holes tape was used to wrap around1-2cycles in order to reinforce drug plaster. Each sticking drug was for4hours x10days;⑤The group of ultrafine herb moxibustion(group B):Application drugs was changed to submicron powder, and the method as above.Experimental observation of targets and testing:①Behavioral observation:After30min and1hour of the last treatment, we observed rats scratching nose, sneezing, experiencing rhinorrhea and other behavioral changes. Scoring method:Nasal itching:mild itching1-2times for1score, intense itching and scratch around the nose for2scores. Sneezing:1-3times for1score,4-10times for2scores, more than11times for3scores. Clear nasal discharge:Flowing to the nostrils for1point, beyond the anterior nostril for2points, and nasal discharge streamed all over the face for3points. We applied the method of superposition scoring.②The nasal mucosa tissue HE staining and observation of eosinophil(EOS) count:After the last treatment, the rats in each group were anesthetized with3ml/kg10%chloral hydrate, then were decapitated to sacrifice. After that we quickly stripped the maxillary skin and separated the maxilla from the skull. Opened the nasal dorsum, and exposed the nasal septum as well as nasal cavity along nasal midline incision. And then, we separated nasal septum and nasal mucosa to take bilateral nasal mucosas which were fixed into10%formaldehyde solution and embedded in paraffin. The routine sections were in hematoxylin-eosin stain. We observed the histological changes of experimental rats’nasal mucosa under an optical microscope. Each of histologic sections randomly selected5high power fields (1040) to count and the number of eosinophils in the nasal mucosa of each rat was as a count.③The TEM (Transmission electron microscope,TEM) observation:Within2minutes, the specimens of nasal mucosa in vitro should be placed in2.5%glutaraldehyde solution and4℃refrigerator overnight. The fixed specimens were flushed with phosphate buffer, and soaked overnight, then fixed1h with1%osmium tetroxide buffer solution and were gradient ethanol dehydration as well as Epon812embedding, polymerization, sliced ultrathin, with uranyl acetate and lead citrate double staining, finally, observed by Hitachi JEM1200-EX transmission electron microscope.④The immunology index determination:using ELISA according to kit instructions to determine the content of IL-4、TGF-β1、IFN-γ、IL-17in serum.Statistical analysis:The behavioral changes were processed with the Kruskal-Wallis of ranked data multiple independent samples of the nonparametric test. Eosinophil count and IFN-γ、IL-4, TGF-β1, IL-17in serum are utilized the single-factor variance analysis. The multiple comparisons were used the method of LSD and SPSS13.0statistical analysis. As result, P<0.05was considered statistically difference.Results:Behavioral observations:①There is significant difference between the scores of model group and the normal control group in nasal itching, sneezing and clear nasal discharge, indicating that AR animal models with ovalbumin sensitization is successfully established;②For the non-parametric tests can not do multiple comparisons, from the mean rank order method all of the three treatments are valid, from the score rating the group B get the best treatment, fluticasone propionate in turn, followed by group A less effective. It means that group B, group A, and beclomethasone dipropionate nasal spray could significantly improve or partly improve the symptoms of allergic rhinitis, including nasal itching, sneezing, clear nasal discharge, symptoms of allergic rhinitis in rats.EOS count results:Compared with the normal control group, significant rise in the model group indicates the successful modeling. The EOS counting of submicron powder group is more significantly decreased (P<0.01) than the model group and group A. Compared with FP group there is also a significantly decrease (P<0.05); while compared with the control group, no significant difference (P=0.539) is observed, which indicates the confirmed local curative effect of group B on treating allergic rhinitis in rats; The EOS counting of FP group and normal powder group were significantly decreased than the model group(P<0.05), compared with the group A, the EOS counting of the FP group significantly decreased with statistical difference (P<0.05), indicating group A treatment of allergic rhinitis not as effective as the nasal hormone.HE staining photographs analysis shows that:Compared with the control group, the model group has significant inflammatory response. angiotelectasis and nasal mucosal epithelium swelling, permeability increase, with a lot of eosinophilic infiltrated lamina propria and surrounding vessels, indicating allergic rhinitis SD rats modeling is successful. The three drug intervened groups compared with the model group are greatly reduced in the inflammation, showing no significant nasal mucosal epithelium swelling, no angiotelectasis and no or a small amount eosinophilic infiltrated lamina propria and surrounding vessels, indicating the treatments of all the drug intervened groups could achieve the desire objectives, which could release the nasal mucous membrane inflammation of allergic rhinitis SD rats. Results of the three groups’photographs are similar to the EOS counting results:FP group and group B were better than the normal group in group A, but HE staining photographs analysis shows no significant difference between the FP group and group B.TEM observations:Inflammatory reaction obviously in the comparison between the model group and normal group, the mucosal surface is not complete, cilia thin and deformity, thick short cilia and microvilli could be seen, part of the cilia fall off, the cilia root incomplete connection in cells, swelling mitochondria and varying sizes vacuoles could be seen in the cells, part of the mitochondria ridge lost, indicating that allergic rhinitis SD rats modeling was successful. Three drug intervened groups achieve more significant improvement than the model group, showing in the mucosal surface is almost complete, the quantity of cilia and microvilli increased significantly, cell junction normal, normal endoplasmic reticulum and mitochondria could even be seen in cells of submicron powder group, which is close to the photograph of the normal control group. Showing that treatment of drug intervened group could reached the desired objective, and allergic rhinitis SD rats nasal mucous membrane of inflammation could be relieved. From the results we could see that the EOS counting is similar to that of HE staining:The curative effects of FP group and group B are better than that of the group A, while the slight difference of normal endoplasmic reticulum and mitochondria could be found in the transmission electron microscope images of group B, which is close to the photograph of the normal control group, better than FP group in curative effect.Serologic observations:Compared with the normal control group, the serum levels of IFN-y model group is dramatically reduced, which indicates the modeling is successful. Compared with the group A and FP group, the serum levels of IFN-y of group B significantly increases (P<0.05), while there is no statistics difference with the comparison between group B and normal control group. Compared with the model group, the content of FP group increases with statistical difference (P<0.05).Compared with the model group, the content of the group A increases with statistical difference (P<0.05), while there is no statistics difference with the comparison between FP group and normal control group (P=0.179). All above shows that:group B, FP and group A treatment of allergic rhinitis of rats significantly increase the abnormal reduce allergy caused by serum IFN-y, with the group B having the best effect and the group A and FP almost the same.The serum levels of IL-4model group compared with the normal control group significantly increases, indicating that the modeling was successful, the submicron powder of IL-4is significantly lower than that of model group, and the group A (P <0.05). Compared with FP group and the normal control group there is no statistically significant difference (P=0.095,0.07). Compared with the model group, content of the FP group decreaseds significantly (P<0.05), group A is statistically significant lower than that of model group (P<0.05), while no significant difference (P=0.396) between the FP with group A, a comprehensive analysis result suggests that:group B, FP for allergic rhinitis treatment in rats of Carcassonne and the group A significantly reduce the abnomal rise of causeserum IL-4caused by allergy, at the same time the similar effect can be found in the group B and FP.Compared with the control group, the model group of TGF-β1increases significantly, suggesting that the modeling was successful. Compared with model group, group A and FP group, the TGF-β1of group B decreases significantly (P <0.05); compared with normal control group there is significant difference (P=0.05); content of the FP group decreases significantly (P<0.05) compared with that of the model group, content of group A compared with that of the model group decreased significantly as well (P<0.05); while there is no statistically significance between the FP group and the group A (P=0.993). a comprehensive analysis result suggests that: group B, FP and group A treatment of rats with allergic rhinitis were significantly reduced allergy caused by serum levels of TGF-β1abnormal rise, while the group B has the best effect, and group A and FP share similar effect.Compared with control group, IL-17model group significantly increases, suggesting that the modeling is successful. Compared with model group, the group B, group A and FP group, IL-17of submicron powder group decreases significantly (P <0.05), and there is no significant difference compared to normal control group(P=0.903). Compared with model group, content of FP group decreased significantly (P <0.05), group A compared with the model group decreased significantly (P<0.05). FP group increases statistically significant (P=0.01) compared with IL-17of group A. A comprehensive analysis result suggests that:the group B, FP and group A treatment of allergic rhinitis in rats reduces the allergic abnormal rise as a result the application of serum IL-17. Also, group B has the best effect, which is followed by intranasal hormone and ordinary powder.CONCLUSION:After the modeling, scores of the AD rat behavioral are all more than5, indicating that intraperitoneal injection ovalbumin (OVA) is reliable AR modeling method. Multiplied the EOS counting, the HE staining, transmission electron microscopy, and serum ELISA results:there were significant differences with the comparison of group B, FP, group A and model group, suggesting that the drug intervention can reduce inflammatory response of AR rats and it is an effective treatment of AR. Although some results are different, the overall trend is consistent with the fact that group B efficacy is superior to FP nasal spray agents and ordinary powder group. Its possible mechanisms are:the stimulations of both medicine and acupuncture points regulate the immune system and reduce the local inflammation. Meanwhile, with the regulatory role of Treg cells, Th cell subsets make the Th1/Th2imbalance relationship return to balance, which promotes Th17differentiation and inhibits proinflammatory function, so that the whole elaborate and complex cells factor networks plays in the balanced and antagonistic immune function. |
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