Establishment of BLM-induced pulmonary fibrosis model in rats
CT is a routine, rapid and non-invasive method for diagnosis and characterisation of IPF.[27] To evaluate the BLM-induced rat model at day 7, CT was used to analysis the marked change in lung. Compared to control rats, the lungs of BLM-treated rats indicated parenchymal opacity, with a main features of a peripheral, predominantly basal pattern of coarse reticulation (Figure 2). Moreover, the CT MLD for BLM-treated rats was -(704.78±19.76) Hu (n=11), and for the control rats the MLD was
-(401.71±22.29) Hu (n=34) (Table 1). ECM molecules including LN, IVC, PIIINP, and HA are the main components of lung collagen and associated with pulmonary fibrosis.[28-30] The serum levels of LN, IVC, PIIINP and HA in BLM-treated rats significantly increased compared with control rats (Table 1). Together with the CT results, it showed that the IPF rat model was well established.
rhCygb reversed BLM-induced histopathological and fibrotic changes in the lung
After the establishment of IPF rat model, the rats were separated into BLM, rhCygb+ BLM (4 mg/kg) and DXMS+ BLM group (3 mg/kg). In figure 3A, CT scan images at day 56 are shown the differences in fibrotic and non-fibrotic lung in BLM-treated and control groups, while rhCygb had improved lung architecture with observable reduced collage deposition and loss of air spaces. Rats received DXMS also had effect on the lungs as compared to that of BLM-treated rats.
Morphologically, compared with the smooth surface of control lung, BLM-treated rats showed the rough, ugly surface of the lung with gray fibrous nodules. On the other hand, rhCygb obviously improved pulmonary morphology in rats with no fibrous nodules (Figure 3B).
H&E staining showed normal lung tissue structure and intact alveolar cavity in the control group. In the BLM-treated group, there was significant alveolar cavity collapse. The alveolar septum, deposition with hemoglobin, was thickened. The alveolar septum was less thickened in the rhCygb+BLM and DXMS+BLM groups (Figure 3C).
Masson trichrome staining (Figure 3D) revealed the lung tissue of the control group had no obvious fibroblast foci. In the BLM-treated group, BLM enhanced pulmonary alveolus inflammation and broadened alveolar septa. Moreover, there was a lot of collagen deposition in lung tissue and the pulmonary fibrosis was severe. In the rhCygb and DXMS treatment groups, the alveolar wall was less thickened and inflammatory cell infiltration decreased. Additionally, there were decreased fibroblasts in lung tissues. However, microthrombus were found in the bronchus of the DXMS treatment group, as compared to control. The Masson CVF results (Figure 3E) showed that the average percentage of lung fibrosis in the control group was 1.86 ± 1.51 %. In contrast, rats treated with BLM resulted in a higher degree of lung fibrosis, which was 40.33 ± 2.08 %. In the rhCygb and DXMS treatment groups, the CVF was 20.81 ± 1.65 % and 30.76 ± 1.57 %, respectively. The comparison between each group was statistically significantly different (Control vs. BLM group, P < 0.001; BLM group vs. rhCygb+BLM group, P < 0.001; BLM group vs. DXMS+BLM group, P < 0.001).
Hydroxyproline levels in lung tissue of BLM-treated group were significantly higher than that in control group (P < 0.05). In the rhCygb and DXMS treatment, HYP levels were obviously lower than that of BLM group (P < 0.05). Moreover, there was significant difference in HYP level between the rhCygb and DXMS groups (P < 0.05) (Figure 3F). This result was consistent with the Masson staining results.
The CT MLD acquired by experiments was shown in Figure 3G. Since most part of the lung is composed by air, the MLD of control lung is tendency towards to -800 Hu. The CT MLD in BLM-treated group was significantly higher than that in control group (P < 0.001). In the rhCygb and DXMS treatment, the CT MLD were obviously lower than that of BLM group (P < 0.001).
Effect of rhCygb on HA, LN, PⅢNP and ⅣC serum levels
The levels of HA, LN, PⅢNP and ⅣC of rats at day 7, 28 and 56 were measured in blood corresponding to the CT analysis in Figure 4. With a longer process of fibrosis, the HA, LN, PⅢNP and ⅣC levels increased from 7 days to 56 days. Serum HA, LN, PⅢNP and ⅣC levels in BLM-treated group were approximately 2.14, 1.61, 1.69, and 1.48 folds of those in the controls, respectively. Both rhCygb and DXMS significantly reduced HA, LN, PⅢNP and ⅣC levels in blood of pulmonary fibrosis rats (P < 0.001). And at day 56, there was no significant difference in HA, LN, PⅢNP levels between rhCygb and control groups except ⅣC levels (P > 0.1).
Correlation of HA, LN, PⅢNP and ⅣC serum levels, HYP and CVF to CT MLD
Positive correlations were observed between CT MLD and LN, IVC, PIIINP, and HA levels, HYP and CVF (Figure 5). The HA, LN, PⅢNP and ⅣC levels exhibited moderate correlations with CT MLD (R2 = 0.4103, P < 0.001; R2 = 0.627, P < 0.001; R2=0.6995, P <0.001; R2 = 0.7489, P <0.001; respectively). HYP also showed a moderate correlation with CT MLD (R2= 0.7127, P <0.001). Masson CVF(%) showed very strong correlations with CT MLD (R2= 0.9376, P < 0.001).