The Physiological Regulation Of Tianlong Extract To Airway Ion Channel And The Significance For Protecting Against Lung Injury Mediated By HA Protein Of H5N1Virus

Background and ObjectiveSevere respiratory diseases caused by SARS coronavirus (SARS-CoV),influenza Avirus subtype H5N1（H5N1）and H1N1pose a major public health threat. Clinical andexperimental studies have shown that pulmonary immunopathology in response to virusinfection is the main reason for high mortality rate in the infected patients, but theunderlying mechanism remains unknown. In preliminary studies, we found that the avianinfluenza virus hemagglutinin（HA）protein can inhibit airway epithelial cystic fibrosistransmembrane conductance regulator (CFTR)function and thereby activate nucleartranscriptional factor (NF-_κB) signaling pathway, contributing to abnormal inflammatoryresponse. So far, no treatment has been shown to be able to overcome this disorder.Traditional Chinese Medicine (TCM), over thousands of years has developed variouspractical theories to create polyherbal formulations in which multiple agents contained inone formula act synergistically. Tianlongkechunling (TLD, developed by a FamousTraditional Chinese Medical Doctor in Guangdong), one such formula mainly composedof Ford nervilia, Tussilago farfara, schisandra chinensis, Monkshood and Pinellia Tuber,has long been used for treating chronic pulmonary disease or respiratory virus infection insouth of China. It has been found that the main active ingredients in TLD formula areflavonoids and other bioactive compounds, which can increase cAMP level to activateCFTR. Based on the findings as above, we hypothesize that TLD might have the potentialbenefits for treating the virus-mediated lung injury through activation of cAMP-dependentCFTR chloride channel. In the present study, we explored the physiological regulation ofthe TLD extract to airway epithelial ion channel by short-circuit current technique. Thepossible mechanism for protection of cAMP-dependent CFTR from the virus-mediateddestruction was investigated.Methods:Part1Physiological regulation of Tianlong extract to airway ion channelTracheas were removed from normal mice (C57BL/6). The tissues were mounted into a perfused micro Using chamber. Transepithelial resistance (Rte) was determined byapplying short (1-s) current pulses and the corresponding changes induced by Tianlongextract in transepithelial voltage (Vte) and basal Vte were recorded continuously.Part2The possible mechanism of TLD extract for protection of cAMP-dependent CFTRfrom H5N1HA-mediated destructionUsing the recombinant H5N1HA protein, the basic short-circuit current of airwayepithelium in the challenged mice was recorded in the absence or presence of TLD extract.The cAMP level of the16HBE cells treated with HA was detected in the absence orpresence of TLD extract, while immunofluorescence was performed for observation ofnucleic localization of NF-_κB.ResultsPart1Activation of Ca2+and cAMP-dependent Cl_ion channels by Tianlong extract inMouse Trachea.Tianlong extract caused an obvious short circuit current (Isc) response in mouse tracheain a concentration-dependent manner. Na channel inhibitor did not block the (Isc)response. Cl-channel inhibitors (DPC,DIDS and glibenclamide), or MDL-12330A andH89were able to partly or totally inhibit the short circuit current (Isc) response. Tianlongcomponents of flavonoids had an apparent weak efficacy for short circuit current (Isc)response.Part2Protection of cAMP-dependent CFTR from H5N1HA-mediated destruction.HA treatment resulted in remarkable reduction at both the basal short-circuit current (Isc)of mice tracheae and cAMP level in the airway epithelial cells, which could be recoveredin the presence of Tianlong. Moreover, addition of forskolin, an agonist of thecAMP-mediated Cl-channel (cystic fibrosis transmembrane conductance regulator, CFTR)increased Isc in the trachea of HA-pretreated mice. Immunofluorescence staining revealedthat nuclear import of NF-_κB was reduced in16HBE cells treated with HA plus Tianlongcompared with HA alone. Conclusions:1.TLD extract can activate the ion channel in airway epithelium resulting in ashort-circuit current response by triggering the airway epithelial CaCC and CFTRCl-channels. And the response was produced by a synergistic action of multi-componentsof Tianlong.2. TLD extract could recover inhibited cAMP dependent CFTR Cl-channels by HAprotein, which contributes to reduction of inflammatory response.