Effects Of Sulfur Dioxide On Biomechanical Behaviors Of The Airway Smooth Muscle From Murine Model Of Asthma

As a chronic respiratory disease, asthma is becoming an increasingly serious threat to human health. Unfortunately, the etiology and development of asthma are still not well understood, while the pathological mechanism of asthma remains difficult to elucidate. As the environment keeps deteriorating, the roles of air pollutants including sulfur dioxide （SO₂） in the pathogenesis of asthma are increasingly interested among investigators. SO₂ is one of the most common air pollutants,and its hazardous effects on respiratory health, especially on asthma has been concerned by many researchers as early as in the last century. Early studies focused on epidemiology, immunology and other aspects, which uncovered various roles of SO₂ in asthma pathology including immunology, oxidative stress and damage, and inflammatory mechanisms. However, there has been no pulished report regarding the effects of SO₂ on biomechanical behavior of the airway smmoth muscle （ASM）. In contrast, there are more and more reports demonstrating that the structure and function of ASM are altered in asthma, which leads to changes of ASM biomechanical behaviors and ultimately results in airway hyperresponsiveness （AHR）. In order to fully understand what does SO₂ do to asthmatics, therefore, it is imperative to study the effects of SO₂ exposure on ASM biomechanical behaviors. Motivated by this question, we established the animal model of asthma using newborn Sprague Dawley （SD） rats that were sensitized by ovalbumin （OVA）. Based on this animal model, we exposed either normal or sensitized SD rats to a 2 ppm （5.6 mg/m3） low dose SO₂ for up to 4 wks, and suqsequently investigated the biomechanical behaviors of the ASM at either organism, tissue, or cell level. The specific and primary findings are described as follows:1) According to the requirement to mimic SO₂ in the environment, we design and build a device that can control the concentration of SO₂ for animal model experiment. This device consists of gas pipeline system, sensor detection systems and intelligent control system, then we can simulate low concentration of SO₂ pollution in the micro-environment with the use of the device, and it has the maneuverability and high reliability properties. After being tested and used for a long period, the result shows that the device can well simulate the single factor of atmospheric SO₂ pollution, animals after being exposed to SO₂ showed significant changes in physiology, all these results proved that this device provided a strong technology platform for investigating the pathogenesis of SD rats, which were exposd to low concentration of SO₂ for a long time.2) The effects of SO₂ exposure on the lung function and airway tissue structure of asthmatic SD rats. First, we used ovalbumin （OVA） as sensitization agent to stimulate the newborn male SD rats, induced airway hyperresponsiveness （AHR）, and took them as the asthmatic animal model for the latter study. On this basis, we used the device talked above to stimulate the normal and asthmatic SD rats with low concentration of SO₂ for a long time, and then observed the lung function （Penh） and airway tissue pathological changes. The results show that, compared with the normal SD rats, after being exposed to SO₂, the values of Penh of asthmatic SD rats increased signicanctly （P<0.01）, which indicates that SO₂ can significantly enhance the hyperresponsiveness of asthmatic SD rats, however, there is no significanct changes for the normal SD rats. Meanwhile, the lung histology hematoxylin-eosin （H&E） staining and the inflammatory factors assay results show that SO₂ can sinnificantly aggravate the inflammatory reaction of asthmatic SD rats.3) The effects of SO₂ exposure on the contractility of airway smooth muscle cells from SD rats. First, we harvested primary airway smooth muscle cells from SD rat successfully by combining the enzymatic digestion method and tissue adherent method, and the immunocytochemistry test proved that the primary airway smooth muscle cells we obtained is relatively high purity, primary cells were passaged to 2-5 generations for the experiments. The results show that this method has the advantages of efficient, fast, easy to operate, low cost, and high volum and purity cells to obtain, and provides sufficient cell sources for the later experiments. On this basis, we measured the biomechanical properties of airway smooth muscle cells by using the optical magnetic twisting cytometry （OMTC）. Airway smooth muscle cells were harvested from normal SD rats （control group）, normal SD rat which were exposed to SO₂（SO₂ group）, asthmatic SD rats（OVA group）, and asthimatic SD rat which were exposed to SO₂（OVA+SO₂ group）. Biomechanical properties including: cell stiffiness, relationship between cell stiffness and frequency, and cell stiffness response to smooth muscle contraction agonist （KCL, histamine）. The results show that, compared with the control group, airway smooth muscle cell stiffness （G0） of the other three groups （OVA+SO₂ group, OVA group, and SO₂ group） was rank ordered and all significantly greater （P<0.001, P<0.001, or P<0.01）, it shows a good power law relationship between cell stiffness and frequency, and there is no significant change in the power-law responses to different frequences. More importantly, among the four groups, only the airway smooth muscle cells from OVA+SO₂ group showed a significant response to postassium chloride （KCL） or histamine.4) The effects of SO₂ derivatives on the biomechanical properties of normal airway smooth muscle cells in vitro. The SO₂ in the atmosphere are ultimately in the form of its derivatives affect the tissues in the body, therefore, on the basis of animal experiments, we investigated different concentrations of SO₂ derivatives （sodium sulfite and sodidum bisulfite, the molar tatio is 3:1） on normal SD rat primary airway smooth muscle cells in vitro. The results show that between 10-4 to 10-1 mmol/L, SO₂ derivatives can enhance the proliferation, migration and contraction of primary airway smooth muscle cells from SD rat, also they can increase the amount of the F-actins, and there exists correlations among the four factors. These results further suggest us a possible pathogenic mechanism which SO₂ worsening the symptoms of asthma can be explained that, by enhancing the proliferation, migration and contraction of airway smooth muscle cells, SO₂ causes remodeling of the airway, hyperresponsiveness, and then in turn exacerbate asthmatic symptoms.In summary, in this paper, we described the biomechanical behavior of airway smooth muscle cells from normal and/or asthmatic SD rats affected by SO₂, respectively, and we studied in individuals, organizations, and celluar level. The results show that, SO₂ exposure can little affect normal SD rats, but it can significantly impact the airway smooth muscle cells from asthmatic SD rats, mainly on the aspects of increasing hyperresponsiveness, the layer of airway smooth muscle, and contractility of airway smooth muscle cells when the asthmatic SD rats were exposured to SO₂. In addition, SO₂ derivatives can enhance the proliferation, migration and contraction of primary airway smooth muscle cells from SD rat cultured in vitro. All these results suggest that, besides of inflammatory response, SO₂ can change the biomechanical behavior of airway smooth muscle cells, especially the response of airway smooth muscle cell to contraction agonist, then increase airway hyperresponsiveness and other typical symptoms of asthma, which providing valuable reference for understanding the role of air pollution in the pathogenesis of asthma on the aspects of biomechanics, also it provides a new way of thinking for improving prevention and treatment of asthma.