Changes Of The Connective Tissue Growth Factor Expression In Mast Cells In Lungs Of Rats In The Development Of Pulmonary Fibrosis And Its Underlying Mechanisms

Objective: Pulmonary fibrosis (PF) is a kind of progressive interstitial diseasein lung, characterised by excessive fibroblast proliferation and collagensdeposition. It is found that the number of mast cells (MCs) was increased inlungs of rats in the development of bleomycin (BLM)-induced fibrosis. Ourprevious studies have found that the number of mast cells was increased andthe connective tissue growth factor (CTGF) was expressed in MCs on serialsections of the lungs of rats on day14and day28after instillation of BLM.However, it needs to be further confirmed. So far, the mechanism by whichCTGF is up-regulated in MCs is still unclear. Tryptase is a specific marker ofMCs. In this study, we firstly confirmed the number of mast cells wasincreased in lungs of rats in the development of pulmonary fibrosis by usingimmunohistochemistry for tryptase, and confirmed CTGF was expressed inMCs by double immunofluorescence histochemistry, then, illuminated themechanism by which MCs were increased and by which CTGF was expressedin MCs in the development of pulmonary fibrosis via aminoguanidine (AG)and baicalin (Bai)Methods: The immunohistochemistry showed the changes of the numberof MCs; the double immunofluorescence histochemistry confirmed theexpression of CTGF in MCs; the HE staining was employed to observe thetypical morphological changes.52Sprague-Dawley (SD) rats(♂)(170-180g) were randomly dividedinto three groups:1. The dynamic observation groups (n=8): Rats were killedto get the lungs on day15and day29after intratracheal instillation of a singledose of BLM (5mg/kg) or a single dose of normal saline (NS,0.5ml/kg) onday14and day28respectively.2. The early treatment groups (1-14d)(n=21): 14dNS+14dNS rats (n=3) were given NS (1ml/kg, i.p) for14days afterintratracheal instillation of a single dose of normal saline (NS,0.5ml/kg). The14dM+14dNS rats:(n=8) were given NS (1ml/kg, i.p) for14days afterintratracheal instillation of a single dose of BLM (5mg/kg). The14dM+14dBai rats (n=5) were given Bai (12.5mg/kg, i.p) for14days afterintratracheal instillation of a single dose of BLM (5mg/kg). The14dM+14dAG rats (n=5) were given AG (20mg/kg, i.p) for14days afterintratracheal instillation of a single dose of BLM (5mg/kg). The above ratswere killed to get the lungs on day15after intratracheal administration.3. Thelate treatment groups (14-28d)(n=23): The28dNS+later14dNS rats (n=3)were given NS (1ml/kg, i.p) from day14to day28after intratrachealinstillation of a single dose of normal saline (NS,0.5ml/kg). The28dM+later14dNS rats (n=8) were given NS (1ml/kg, i.p) from day14to day28afterintratracheal instillation of a single dose of BLM (5mg/kg). The28dM+later14dBai rats (n=6) were given Bai (12.5mg/kg, i.p) from day14to day28afterintratracheal instillation of a single dose of BLM (5mg/kg).The28dM+later14dAG rats (n=6) were given AG (20mg/kg, i.p) from day14to day28afterintratracheal instillation of a single dose of BLM (5mg/kg). The above ratswere killed to get the lungs on day29after intratracheal administration. Therats in each group were killed at a specified time point and their lung tissueswere used for detecting the number of MCs and CTGF immuno-positive MCs,as well as for observing the morphological changes in lung.Using artificial notation, five high-magnification non-overlaping fileds ofvision were randomly selected for each immunohistochemistry slice toaccurately count the number of MCs, and three slices of the different parts ofthe lung tissue of each rat were observed to calculate the average of MCs.Three low-magnification non-overlaping fileds of vision were randomlyselected for each immunofluorescence staining slice to accurately count thenumber of MCs and CTGF immuno-positive MCs, and three slices of thedifferent parts of the lung tissue of each rat were observed to calculate theaverage of MCs and CTGF immuno-positive MCs. All data were shown by mean±standed deviation. After homogeneitic analysis, homogeneous datawere analyzed with one-way ANOVE analysis of variance and a post hoc testof least significant difference (LSD) by using SPSS-13.0statistical software. Pvalues less than0.05was viewed as statistically significant.Results:1Changes of the number of MCs and CTGF immuno-positive MCs in lungs ofrats.1.1Dynamic changes of the number of the MCs: Immunohistochemicalstaining and immunofluorescence staining showed: Compared with control rats,the number of MCs was increased in BLM rats on day14and28(P<0.01,P<0.001). Compared with14dM rats, the changes of the number of MCs in28dM rats were of no statistical significance (P>0.05).1.2Dynamic changes of the number of CTGF immuno-positive MCs:Compared with control rats, the number of CTGF immuno-positive MCs wasincreased in BLM rats on day14and28(P<0.001). Compared with14dM rats,the changes of the number of CTGF immuno-positive MCs in28dM rats wereof no statistical significance (P>0.05). Compared with control rats, the ratio ofCTGF immuno-positive MCs to pulmonary MCs in14dM and28dM rats wasof no statistical significance (P>0.05). Compared with14dM rats, the changesof the ratio of CTGF immuno-positive MCs to pulmonary MCs in28dM ratswas of no statistical significance (P>0.05).2Effects of AG and Bai on the number of MCs and CTGF immuno-positiveMCs at the early phase (1-14d) of pulmonary fibrosis.2.1Effects of AG and Bai on the number of MCs at the early phase ofpulmonary fibrosis: Immunohistochemical staining and immunofluorescencestaining showed: Compared with14dNS+14dNS rats, the number of MCs in14dM+14dNS rats was enhanced significantly (P<0.001). Compared with14dM+14dNS rats, the number of MCs in14dM+14dAG and14dM+14dBairats was decreased significantly (P<0.001, P<0.01).2.2Effects of AG and Bai on the number of CTGF immuno-positive MCs atthe early phase of pulmonary fibrosis: Compared with14dNS+14dNS rats, the number of CTGF immuno-positive MCs in14dM+14dNS rats was enhancedsignificantly (P<0.01). Compared with14dM+14dNS rats, the number ofCTGF immuno-positive MCs in14dM+14dAG and14dM+14dBai rats wasdecreased (P<0.01). Compared with14dNS+14dNS rats, the ratio of CTGFimmuno-positive MCs to pulmonary MCs in14dM+14dNS rats was of nostatistical significance (P>0.05). Compared with14dM+14dNS rats, the ratioof CTGF immuno-positive MCs to pulmonary MCs in14dM+14dAG and14dM+14dBai rats was of no statistical significance (P>0.05).3Effects of AG and Bai on the number of MCs and CTGF immuno-positiveMCs at the late (14-28d) phase of pulmonary fibrosis3.1Effects of AG and Bai on the number of MCs at the late phase ofpulmonary fibrosis: Immunohistochemical staining and immunofluorescencestaining showed: Compared with28dNS+later14dNS rats, the number ofMCs in28dM+later14dNS rats was enhanced significantly (P<0.001).Compared with28dM+later14dNS rats, the number of MCs in28dM+later14dAG and28dM+later14dBai rats was decreased significantly (P<0.001,P<0.01).3.2Effects of AG and Bai on the number of CTGF immuno-positive MCs atthe late phase of pulmonary fibrosis: Compared with28dNS+later14dNS rats,the number of CTGF immuno-positive MCs in28dM+later14dNS rats wasenhanced significantly (P<0.01). Compared with28dM+later14dNS rats, thenumber of CTGF immuno-positive MCs in28dM+later14dAG and28dM+later14dBai rats was decreased (P<0.01). Compared with28dNS+later14dNSrats, the ratio of CTGF immune-positive MCs to pulmonary MCs in28dM+later14dNS rats was enhanced significantly (P<0.05). Compared with28dM+later14dNS rats, the ratio of CTGF immuno-positive MCs to pulmonary MCsin28dM+later14dAG and28dM+later14dBai rats was of no statisticalsignificance (P>0.05).4Effects of AG and Bai on the typical morphology (HE staining) at the earlyand late phases of pulmonary fibrosis.4.1Changes of morphology in lungs of rats in BLM-induced fibrosis: The lung structure of the14dNS and28dNS rats was normal. But in14dM and28dMrats, the alveolar septum was obviously enlarged, with increased inflammatorycells.4.2Changes of morphology in lungs of rats at the early phase of pulmonaryfibrosis: The lung structure of the14dNS+14dNS rats was normal. Thealveolar septum in14dM+14dNS rats was obviously enlarged. The abovemorphological changes in14dM+14dNS rats were obviously improved in the14dM+14dAG and14dM+14dBai rats.4.3Changes of morphology in lungs of rats at the late phase of pulmonaryfibrosis: The lung structure of the28dNS+later14dNS rats was nomal. Thealveolar septum in28dM+later14dNS rats was obviously enlarged. In28dM+later14dNS rats, mesenchymal cell infiltration existed in lung. The abovemorphological changes were obviously improved in the28dM+later14dAGand28dM+later14dBai rats.Conclusion:1The number of MCs was increased and CTGF was expressed in MCs in thedevelopment of pulmonary fibrosis2At the early and late phase of BLM-induced pulmonary fibrosis, AG and Baicould reduce the number of MCs and CTGF immuno-positive MCs. Thisimplied that AG down-regulated the content of NO and Bai lipid peroxidationdamage to reduce the number of MCs and CTGF immuno-positive MCs.