Effects Of N-acetylcysteine On Bleomycin-induced Lung Injury And Pulmonary Fibroblast From NSIP Patient

PARTⅠAttenuation by oral N-acetylcysteine of bleomycin- induced lung injury in ratsObjective: To study the role of oral N-acetylcysteine (NAC) in treatment of bleomycin-induced lung injury in rats.Methods: All 240 SD rats were randomly divided into 6 groups: normal control group, model group(bleomycin challenge alone), NAC low-dose group, NAC high-dose group, prednisone group and prednisone combined with NAC group. The animals were satisfied respectively at 3,7,14 and 28 days after intratracheal injection of bleomycin (5 mg/kg) respectively. The lung sections were prepared for pathological examination. The basic-fibroblast growth factor (bFGF) and the platelet-derived growth factor BB (PDGF-BB) in bronchoalveolar lavage fluid (BALF) were tested by enzyme-linked immunosorbent assay (ELISA).Results: Pathological examination demonstrated that bleomycin resulted in a severe fibrotic injury at 14 days after the bleomycin injection in the rats of model group. Compared to the rats of the model group, NAC high, NAC low dose group, prednisone group as well as combined group had significantly lower Ashcroft scores (P <0.05). Compared with the normal group, the hydroxyproline content in lung tissue was significantly increased (P <0.01) in the model group whereas the treatment of either high-dose NAC or prednisone or combined theraputic attenuated production of hydroxyproline induced by the bleomycin injection. At 28 days after the bleomycin injection, Ashcroft scores returned to normal in the rats of prednisone group or the combined group while the rats of NAC high dose or low dose group had a higher Ashcroft score than the rats of control normal. Three days after the intratracheal instillation of bleomycin the bFGF level in BALF was significantly increased from (41.60±20.49) pg/ml to (212.69±55.11) pg/ml, (P = 0.001), and returned to normal at the seventh day. Treatment with NAC 500mg.kg-1.d held back the peak. At 28 days after the bleomycin injection, bFGF level in BALF had no difference among all groups. The changes of levels of PDGF-BB and bFGF in BALF were the same.Conclusion:1. The treatment with NAC in the early phase attenuated the bleomycin- induced lung injury in rats.2. The anti-fibrosis efficacy of NAC may be ascribed to its anti-inflammatory and down-regulation of growth factor PDGF induction by bleomycin in the lung of the rats.3. Combination of NAC with prednisone may not have an anti-fibrotic synergie.PARTⅡEffects of NAC's action on fibroblasts derived from NSIP patient and the normal.Section 1. Effects of NAC on proliferation of the pulmonary fibroblast derived from NSIP patient and the normal.Objective: To study effects of NAC on cell growth of the pulmonary fibroblasts derived from NSIP patient and the normal.Methods: Primary lung fibroblasts were prepared from the pulmonary biopsy of a NSIP patient and uninvolved adjacent lung tissues obtained from patients with lung cancer during surgical operation. Intervention of NAC on proliferation of the fibroblasts either from NSIP or normal lung tissues was evaluated.While typeⅡalveolar epithelial cells A549 were also subjected to the examination. Results:①NAC (20 mM) caused a significant growth inhibition in the normal lung fibroblast (HFB), but not in alveolar epithelial cell line A549.②NAC at different concerntrations (5~40Mm) can inhibit the proliferation of pulmonary fibroblasts either of NSIP and normal.③NAC at different concerntrations (5~40Mm) can also decrease the expression of procollagen typeⅠgene in HFBs under basal condition as well as attenuate the induction of procollagen typeⅠgene expression by TGFβ1..Conclusion:1. NAC shows a cell-specific anti-proliferation on fibroblasts in a concentration-dependent model.2. The pulmonary fibroblasts derived from NSIP demonstrate the drug-resistance of growth inhibition in response to NAC.3. NAC can also down-regulate typeⅠprocollagen synthesis in pulmonary fibroblasts in the absence or presence of TGFβ1.Section 2. Comparison of regulation ofα-SMA expression by TGF-β1 signaling in the pulmonary fibroblasts between NSIP and normal lung in response to N-acetylcysteine(NAC)Objective: To study the regulation ofα-SMA expression by TGF-β1 signaling in the pulmonary fibroblasts of NSIP and normal lung in response to N-acetylcysteine.Methods: The primary pulmonary fibroblasts of NSIP and normal were cultured as above and four to six- passage cells were used for the experiment. The cells were divided into 4 groups: control, NAC group, TGF-βgroup and TGF-βplus NAC group. Western blotting was performed on detecting expression ofα-SMA and STAT3 in the both pulmonary fibroblasts with different treatments.Results:①The expression ofα-SMA protein in normal lung fibroblasts was enhanced by TGF-β1 stimulation and was able to be slightly inhibited by NAC. There were no significant differences among control, NAC and TGF-βgroups.②The expression ofα-SMA is higher significantly in the pulmonary fibroblasts of NSIP- lung than that in those of normal lung. NAC increasedα-SMA expression significantly related to up-regulation of STAT3 expression in the pulmonary fibroblasts of NSIP lung. While TGFβ1 can inhibitα-SMA expression related to down-regulation of STAT3 expression in the NSIP- fibroblasts. The pulmonary fibroblasts of NSIP lung express phosphoralyed STAT3 under basal condition but not in those of normal lung. TGFβ1 can inhibit the expression of phosphoralyed STAT3 in the NSIP-fibroblasts.Conclusion:1. Pulmonary fibroblasts from NSIP patient increased expression ofα-SMA, non- phosphoralyed STAT3 and phosphoralyed STAT3 signal molecular compared to normal cells.2. NAC can induce the expression ofα-SMA related to up-regulation of STAT3 signal molecular in the pulmonary fibroblasts from NSIP patient.3. TGF-βcan inhibit the expression ofα-SMA related to down-regulation of non-phosphoralyed and phosphoralyed STAT3 in the pulmonary fibroblasts from NSIP patient.Section.3. Effects of NAC on levels of chemokines /cytokines released from pulmonary fibroblasts of NSIP patient's and normal lungsObejective:To investigate effects of NAC on levels of chemokines /cytokines released from pulmonary fibroblasts of NSIP patient's and normal lungs in the absence or presence of TGF-β1. Methods: The cultured pulmonary fibroblasts of NSIP and normal lungs were divided into 4 groups: control, NAC group, TGF-β1 group and TGF-β1 plus NAC group. Supernatants from those cells cultured with the various treatments in the different groups were collected for measurement of levels of chemokines /cytokines by liquid-chip assay with a Luminex.Results:①The pulmonary fibroblasts of NSIP-lung produced higher levels of chemokines /cytokines than normal cells such as MIP-1α, MIP-1β, MIP-3α, MIG, MCP-3, VCAM-1, TNF-α, TGF-β, ENA-78, MMP-9 and IL-18 under basal condition.②TGF-β1 increased the levels of chemokines /cytokines released from either of the both cells, The pulmonary fibroblasts of NSIP-lung had an ability to increase production of chemokines /cytokines after exposed to TGF-β1.③NAC attenuated the induction of chemokines /cytokines in the normal cells, but not in the NSIP-lung fibroblasts.④There was a lower level of IP-10 in the supernatant of the NSIP-lung fibroblasts than that of the normal cells under basal condition. TGF-β1 increased the level of IP-10 in the supernatant of NSIP-lung fibroblasts whereas NAC inhibited the basal IP-10 expression in those cells.Conclusion:1. The pulmonary fibroblast from NSIP patient exhibits different biological characteristic from the normal.2. The pulmonary fibroblasts from NSIP patient have an ability to produce high levels of chemokines /cytokines with or without TGF-β1 stimulation compared to normal lung fibroblasts.3. NAC can attenuate the basal expression of some of chemokines /cytokines in the NSIP-lung fibroblasts but not the induction of chemokines /cytokines by TGF-β1, which is associated with an abnormal TGF-β1-receptors- signaling...