| Bronchial asthma is a frequently-occurring disease.It is of great difficulty to cure. The chronic airway inflammation is caused by eosinophils, mast cells and T lymphocytes and other inflammatory cells.This inflammation causes airway hyper-increased reactivity and the airways to narrow.The clinical manifestations are symptoms such as recurrent episodes of wheezing, dyspnea, chest tightness or cough. It usually attack or intensified at night and (or) early morning. There are lots of widely varied reversible airflow limit. The majority of patients could spontaneously relieved or after treatment. In the traditional Chinese medicine we call it" Asthma "," sipping cough "," croup " and so on. The disease is not caused by a single facror. Its pathogenesis is extremely complex, and yet entirely not clear, which is mainly related to airway inflammation, airway hyperresponsiveness and airway remodeling. It has reached a consensus that asthma is a non-specific inflammatory airways disease. The international study of airway remodeling has already put into agenda. Structural changes in airway remodeling result in airway hyperresponsiveness (AHR), persistent airflow obstruction and irreversible lung damage. Currently found in transforming growth factor β1(TGF-β1)/Smad signaling pathway is an important signal transduction mechanisms which lead to the formation of airway remodeling.The studies have shown that TGF-β1in airway (?)emodeling plays an important role in the expression of airway basement (?)embrane thickness and the number of fibroblasts which is positively (?)orrelated to the severity of asthma.The back three-ponit electrical stimulation may reduce the protein (?)xpression of TGF-betal in the bronchopulmonary tissue of the airway remodeling guinea pig model. The Smad3protein are involved in regulation of TGF-β1signal transduction.It plays an important role in promoting the expression of TGF-β1biological activity.And thereby it promotes the development of remodeling.We choose the Smad3as downstream signal-regulated protein of TGF-beta/Smad signal path. We study acupuncture for airway remodeling in the regulation of this signaling pathway to explore molecular mechanism of the acupuncture treatment of bronchial asthma. It provides in-depth theoretical basis for its clinical application promotion.Purpose:By observing electropuncture on the back three acupoints(Dazhu, Fengmen, Feishu) on airway remodeling in asthmatic rats model transforming growth factor-β1(TGF-β1) and Smad3protein and mRNA expression of airway remodeling in asthmatic rats model, we could explore molecular mechanisms of EA back three ponits treatment on bronchial asthma.Method:The44SPF male SD rats were randomly grouped into EA group (A)12, dexamethasone group (B)12, model group (C)10and control group (D)10rats. In addition to the control group without any intervention, the rest of the rats are intervented by lml of OVA (ovalbumin)with aluminum hydroxide adjuvant intraperitoneal injection, which contains ovalbumin100mg and aluminum hydroxide100mg. The sensitized progress is twice, a period of two weeks. At the15th day of the experiment, we begin to supply1%OVA inhalation to excite the rats in a period of six weeks, in order to establish asthma model. The dexamethasone group is given dexamethasone therapy at0.5mg/kg intraperitoneal injection half an hour before each atomization.The electro-acupuncture group is performed on back three ponits Dazhu, Fengmen and Feishu before the atomization excited. The three ponits are located at7mm adjacent to the first, second third thoracic vertebra.The acupuncture direction is45°direction obliquely to the head in the direction of the spine, at a depth of about3mm with twisting the needle after needle after about1minute.And then the group is given a current stimulation waveform selection density wave, with the frequency is16beats/min, for15minutes. The EA strength is to be able to withstand in rats and when our hands touch the needle we can feel the beat for the degree of treatment every other day. The total test are eight weeks. At the end of the last treatment interventions, each group were weighed. According to the weight, each rat is given10%3.5ml/kg dose intraperitoneal injection of chloral hydrate anesthesia. After skin disinfection,we opened the chest and did cardiac perfusion, took the left lung tissue, with4℃saline rinse immediately and then placed it in4%paraformaldehyde solution more than24hours. After embedded in paraffin and sliced, it was given HE staining and immunohistochemical detection. Using the image analysis software to detect muscle’s standardized cross-sectional area and the bronchial wall’ s standardized cross-sectional area. Furthermore, other lung tissue was put into2ml vials of blood after rinsing with4℃saline, and was placed in-70℃cryopreservation. It was prepared to do the real-time PCR detection of TGF-β1and Smad3gene expression levelsResults:1.General observation:After OVA inhalation excited every time, model group show poor spirit, nodding motion, rapid breathing, occasional sneezing, showing irritability, scratching, urine and other symptoms. The electropuncture group and the dexamethasone group with mild symptoms after atomization also appears to stimulate fur light chromaticity fall loose, respiratory rate, sneezing and nodding movement. But the dexamethasone group’s weight increase is not obvious. They looked emaciation.2. The pathological changes in the morphology of lung tissueBronchial epithelial cells in model group appears to fall off and the number of goblet cells increase. There are mucosal edema, airway mucosa, submucosa and perivascular tissue inflammatory cell infiltration, such as eosinophils, lymphocytes and plasma cells. There are also smooth muscle thickening and stenosis occlusion. Although there are mucosal edema, inflammatory cell infiltration, smooth muscle thickening in the EA group and dexamethasone group, the degree is relatively light.3. The morphology index changes in lung tissue under an optical microscope:According to the results analysis of the image acquisition software, compared with the control group, the standardized muscle cross-sectional area (WAm/Pbm)and the standardized bronchial wall cross-section area (WAi/Pbm)of the EA group and the dexamethasone group and model group are improved with statistical significance (P<0.05). Compared with the model group, the EA group and the dexamethasone group are lower with statistical significance (P<0.05). There are no statistical significance (P>0.05)between the electropuncture group and the dexamethasone group.4. The protein expression of TGF-β1in lung tissue:According to immunohistochemical results, compared with the control group, the TGF-β1protein expression levels of the electropuncture group,the dexamethasone group and the model group are improved with statistical significance (P<0.05). Compared with the model group, the protein expression of the EA group and the dexamethasone group are lower with statistical significance (P<0.05).There are no statistical significance (P>0.05)between the electropuncture group and the dexamethasone group.5. The gene expression of TGF-β1in lung tissue:According to the results of qRT-PCR, we could learn that the mRNA expression of TGF-β1in model group, the EA group and the dexamethasone group significant difference in the relative expression volume has improved with statistical significance(P<0.05), compared with the control group making the blank group as reference. Compared with the model group, the TGF-β1mRNA are lower in the EA group and the dexamethasone group with statistically significant (P<0.05). There are no statistical significance (p>0.05)between the electropuncture group and the dexamethasone group.6. The expression of the Smad3protein that is the signal transduction downstream of the lung key regulator:According to immunohistochemical results, compared with the control group, Smad3protein expression levels of electro-acupuncture group, dexamethasone group and model group had improved with statistical significance (P<0.05). Compared with the model group, the protein expression were decreased between the acupuncture group and the dexamethasone group, with statistical significance (P<0.05). There are no statistical significance(P>0.05)between the electropuncture group and the dexamethasone group. 7. The gene expression of Smad3in lung tissue:According to the results of qRT-PCR, we learn that the mRNA expression in the model group, the eletropuncture group and the dexamethasone group significantly difference(P<0.05) compared with the control group making the blank group as reference. Compared with the model group, the Smad3mRNA are lower in the EA group and the dexamethasone group, and there is statistically significant (p<0.05). There are no statistical significance(p>0.05) between the electropuncture group and the dexamethasone group.Conclusion:1.Compared with the control group, airway morphological parameters of the model group-the standardized muscle cross-sectional area and the standardized cross-sectional area of the bronchial wall are increased. There is statistically significant, indicating successful model.After the treatments, both of the areas are decreased, with statistical significance, indicating that the two treatments reduce the airway morphological parameter and the degree of airway remodeling. But the EA group and the dexamethasone group and the control group are still statistically significant, indicating the extent of the two treatment methods are not able to cause airway remodeling return to normal. Meanwhile the EA group and the dexamethasone group have similar effect, there is no statistically significance after contrast.2. Compared to the control group, the expression of TGF-β1protein and mRNA of the model group are both increased but decreased in the electro-acupuncture group and dexamethasone group but after being treated. It indicates that these two treatments could reduce the expression of TGF-β1. Thereby it could inhibiting the signal transduction pathway and airway remodeling effect. But the EA group and the dexamethasone group and the control group still have statistically significance, indicating that these two interventions have failed to reduce to the scope of the normal rats. Meanwhile, there are no statistically significance between the electro-acupuncture group and the dexamethasone group. It indicates that the two methods could achieve a similar therapeutic effect.3. Compared to the control group, the expression of Smad3protein and mRNA are enhanced in the model group. After being treated, the Smad3protein and mRNA expression are reduced in the electro-acupuncture group and the dexamethasone group, indicating that these two treatments could reduce the expression of Smad3, thereby inhibiting the signal transduction pathway and airway remodeling.But the EA group and the dexamethasone group and blank group still have statistically significance, indicating that these two interventions have failed to reduce to the scope of the normal rats Meanwhile, the electro-acupuncture group and the dexamethasone group have no statistically significance, indicating that the two methods can achieve a similar therapeutic effect.To sum up, the results show that electroacupuncture on back three ponits could reduce the TGF-β1and the Smad3protein and gene expression levels.And it inhibit the TGF-β1/Smad3signaling pathway, in order to reduce the airway remodeling and relieve asthma symptoms... |
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