## 3 Results
### Effect of CBD on Gene Expression Associated with Pulmonary Fibrosis  
RT-PCR analysis of the pulmonary expression of TGF-\(\beta\) (\(p<0.001\)), \(\alpha\)-SMA (\(p=0.009\)), MMP-7 (\(p<0.001\)), MMP-9 (\(p<0.001\)), and TIMP-1 (\(p=0.011\)) mRNAs showed that exposure to CS and LPS (COPD group) induced significant increases in the expression of these genes in lung tissues, compared with saline-exposed mice (control group) (Figure 5). Treatment with RO (TGF-\(\beta\): \(p=0.009\), \(\alpha\)-SMA: \(p<0.001\), MMP-7: \(p<0.001\), MMP-9: \(p=0.023\), and TIMP-1: \(p=0.007\)), CBD 100 (TGF-\(\beta\): \(p<0.001\), \(\alpha\)-SMA: \(p<0.001\), MMP-7: \(p=0.019\), MMP-9: \(p=0.015\), and TIMP-1: \(p=0.035\)), or CBD 200 (TGF-\(\beta\): \(p<0.001\), \(\alpha\)-SMA: \(p<0.001\), MMP-7:  
Figure 3: Effects of CBD on concentrations of proinflammatory cytokines in BALF. Individual samples were analyzed using ELISA. (**A**) IL-6. (**B**) TNF-\(\alpha\). Control: mice instilled intratracheally with normal saline; COPD: mice exposed to CS and LPS; RO: mice exposed to 10 mg/kg rofumilast plus CS and LPS; CBD 100: mice exposed to 100 mg/kg CBD plus CS and LPS; CBD 200: mice exposed to 200 mg/kg CBD plus CS and LPS. Results are presented as means \(\pm\) SEMs. \({}^{\#}\)\(p<0.01\) compared with the control group; \({}^{*}\)\(p<0.05\), \({}^{**}\)\(p<0.01\) compared with the COPD group.  
Figure 2: Effects of CBD on inflammatory cell recruitment to COPD-induced mice BALF. (**A**) Number of total cells. (**B**) Number of lymphocytes. (**C**) Number of macrophages. Control: mice instilled intratracheally with normal saline; COPD: mice exposed to CS and LPS; RO: mice exposed to 10 mg/kg rofumilast plus CS and LPS; CBD 100: mice exposed to 100 mg/kg CBD plus CS and LPS; CBD 200: mice exposed to 200 mg/kg CBD plus CS and LPS. Results are presented as means \(\pm\) SEMs. \({}^{\#}\)\(p<0.01\) compared with the control group; \({}^{*}\)\(p<0.05\), \({}^{**}\)\(p<0.01\) compared with the control group.  
\(p<0.001\), MMP-9: \(p=0.013\), and TIMP-1: \(p=0.013\)), however, significantly attenuated the COPD-induced increases in these mRNA levels (Figure 5).  
Figure 4: Analysis of mRNA levels of inflammatory cytokines in lung tissues. Expression of (**A**) IL-18, (**B**) IL-6, (**C**) TNF-\(\alpha\), and (**D**) MCP-1 mRNAs was measured by RT-PCR. Control: mice instilled intratracheally with normal saline; COPD: mice exposed to CS and LPS; RO: mice exposed to 10 mg/kg rofumilast plus CS and LPS; CBD 100: mice exposed to 100 mg/kg CBD plus CS and LPS; CBD 200: mice exposed to 200 mg/kg CBD plus CS and LPS. Results are presented as means \(\pm\) SEMs. \({}^{\#}\)\(p<0.05\), \({}^{\#}\)\(p<0.01\) compared with the control group; \({}^{\star}\)\(p<0.05\), \({}^{\#}\)\(p<0.01\) compared with the control group; \({}^{\star}\)\(p<0.05\), \({}^{\#}\)\(p<0.01\) compared with the COPD group.  
Figure 5: Effects of CBD on levels of mRNAs encoding pulmonary-fibrosis-related genes in lung tissues. Levels of (**A**) TGF-\(\beta\), (**B**) \(\alpha\)-SMA, (**C**) MMP-7, (**D**) MMP-9, and (**E**) TIMP-1 mRNAs were measured by RT-PCR. Control: mice instilled intratracheally with normal saline; COPD: mice exposed to CS and LPS; RC: mice exposed to 10 mg/kg rofumilast plus CS and LPS; CBD 100: mice exposed to 100 mg/kg CBD plus CS and LPS; CBD 200: mice exposed to 200 mg/kg CBD plus CS and LPS. Results are presented as means \(\pm\) SEMs. \({}^{\#}\)\(p<0.05\), \({}^{\#}\)\(p<0.01\) compared with the control group; \({}^{\star}\)\(p<0.05\), \({}^{\#}\)\(p<0.01\) compared with the COPD group.