Studies On The Activation Of VR1 By Formaldehyde And Molecular Mechanisms Of Formaldehyde Induced Asthma

In recent years, concern about the indoor air pollutant and its health effects has been arisen. The formaldehyde induced airway neurogenic inflammation, acquired atopy and so on, involved in the pathological process of the bio-target molecule of formaldehyde. In 1991, G. D. Nielsen, an environmental medicine scientist, first advanced a hypothesis: indoor air pollutants (e.g. formaldehyde) induced airway irritation is a receptor-mediated pathological process, and the receptor is capsaicin receptor. Due to the lack of experimental supporting, the hypothesis had become an unsettled case from then on. It is confirmed that capsaicin receptor is VRl. VR1~+ cell model is a biological active cell that constructed from VRl (vanilloid receptor subtype 1) membrane protein and culture cell line in vitro. It was first constructed at California University in 1997 and it was applied in studies on mechanism of pain pathway and molecular pharmacology. The construction of VR1~+ cell model could provide a research strategy to verify this hypothesis. If the study progress of VRl applied into environmental medicine, it may promote the mechanism research on indoor air pollution and human diseases, such as formaldehyde induced asthma.The present project studied the activation of VRl by formaldehyde through experimental study in vitro and in vivo, to elucidate the mechanism of bio-target molecule of formaldehyde and verify the Nielsen's hypothesis; we also studied the molecular mechanisms of formaldehyde induced asthma, the results are as follows:1. Construction of VR1~+-HEK293 cell model and verification ofNielsen's hypothesisTo explore the physiological role of vanilloid receptor type 1 (VRl) as a possible bio-target molecule of indoor-air pollutant, formaldehyde, VR1~+-HEK293 cell model was constructed using the VR1-cDNA plasmid through the molecular cloning and transfection technology. The experiments of capsaicin and formaldehyde induced death of transfected VR1~+-HEK293 cells were carried out. The Results showed that VR1~+-HEK293 cell model was constructed successfully, and measurement ofVR1+-HEK293 cell death rate showed to be an available and effective method, which can be applied to detecting the VR1 expression in cells in vitro; activated VR1 by formaldehyde could be blocked by capsazepine, a specific antagonist of VR1, it was suggested that the irritant effect of airway may be also mediated by VR1. Therefore, it is concluded that VR1 as a possible bio-target molecule of formaldehyde, i.e. Nielsen's hypothesis is correct according to our experiments in vitro.2. The voltage clamp study on expression of VR1 in Xenopus oocytesVR1+ model cell, constructed by David Julius's group in 1997, made the systematic study using cell expression system in vitro on VR1 possible. Whether formaldehyde can be a ligand to activate VR1, and mediate neurogenic inflammation via type I VR1 signaling system (FA/VRl/Ca2+/SP/NKR), and mediate the development of acquired atopy via type II VR1 signaling system (FA/VRl/Ca2+/IL-4/IgE)? Whether other indoor air pollutants, such as PM2.5, PAEs can mediate the toxicological effects by the activation of VR1? To answer these questions, the voltage clamp study provided an effective detection method.Due to the difficulty and complexity of electrophysiology recording technology in the whole study, we conducted endogenous ATP-induced currents experiment on Xenopus oocytes. On the samples of Xenopus oocytes, three different kinds of electrical current were recorded, which is consistent with the results reported by Professor Li Zhiwang's group, indicating the success of our electrophysiology recording. By far, we have finished transcription in vitro, microinjection and Xenopus oocytes culture in vitro. It is expected to get expressed VR1+ Xenopus oocytes, and to verify the activation of VR1 by formaldehyde through the voltage clamp technique.3. The activation of cutaneous VR1 in mice by formalin ofnon-inflammatory doseTo explore the relations between the activation of vanilloid receptor type 1(VR1) with cutaneous formalin stimulation of non-inflammatory dose in mice. Male KM mice were randomly divided into 3 groups: FM groups, Capsaicin (CAP) groups and Capsazepine(CPZ) groups. The amount of time spent licking the injected paw within 5min after injection was timed with a chronometer and was considered as an index ofpain. The results showed that 200^mol/L CAP and 0.025% FM were optimal concentrations of the pain reagent, which could evoke a painful sensation but not cause inflammation. l^mol/L CPZ had obvious anti-nociceptive effects to the CAP- and FM-induced pain. In conclusion, pain induced by non-inflammatory dose of FM in peripheral was possibly mediated by VR1.4. Acquired atopy: a possible mechanism of formaldehyde inducedasthmaThe prevalence of asthma keeps on increasing worldwide, especially in western societies. But the mechanism of asthma is unclear. Recently, concern about indoor air pollution as a risk factor for asthma has been arisen. In present study, 25 BALB/c male mice were placed in an air chamber containing respective gaseous formaldehyde (FA) concentration of 0, 0.5, 1.0, 3.0mg/m3 and 3.0mg/m3 with capsazepine (CPZ, a specific antagonist of VRl)-pretreatment in five testing groups (n=5 per group) for inhale experiments. The inhaled groups were exposed to gaseous FA for 6 hours each day in 10 successive days. After exposure, the concentrations of IL-4 in broncho alveolar lavage fluid (BALF) and total IgE in blood serum were measured. The results showed that all the IL-4 level in BALF and total IgE level in serum increased in a dose-dependent manner. However, for the CPZ-pretreated group the ILr4 and total IgE level decreased significantly (compared with 3.0mg/m3 FA inhaled group, p<0.01). The results indicated that gaseous FA might induce acquired atopy by type II VR1 signaling system. These findings suggested that indoor air pollutants such as FA might be key risk factors for the rise in asthma cases, and type IIVR1 signaling system might be one of the mechanisms for the rise.5. GSNOR pathway: a possible mechanism of formaldehyde-inducedasthmaS-nitrosoglutathione reductase (GSNOR) is an important enzyme that has drawn great attention in recent years, because of its capacity of metabolism S-nitrosoglutathione (GSNO), which is an endogenous bronchodilator versus active constriction. Recent study showed that the interaction of NO and "active NO" (GSNO) possesses a crucial role in airway physiology and pathophysiology. GSNOR regulates the equilibrium of NO and GSNO, and they are involved in the development of airway diseases, e.g., asthma.Interestingly, it has been noticed that GSNOR is formaldehyde dehydrogenase (FDH), an enzyme that is important in formaldehyde metabolism. We proposed that inhaled formaldehyde may up-regulate GSNOR expression, deplete GSNO concentration in the airways consequently, and result in the increase of smooth muscle tone. With other allergen, asthma may occur in patients with decreased GSNO levels. In our study, 18 male KM mice were divided randomly into 3 groups, and were exposed to 0, 1.0, 3.0 mg/m3 gaseous formaldehyde for 72h continuously. RT-PCR was applied to examine the transcription of GSNOR mRNA in mice lungs, and results showed that inhaled formaldehyde can induce the up-regulation of GSNOR expression, and metabolize GSNO consequently, which is consistent with the conclusion that inhaled formaldehyde (above 1.0 mg/m3) can result in the increase of NO level in the lung, indicating the equilibrium of NO and GSNO in the mediation of airway physiology and pathophysiology.