Fig 2: RAAV-mediated SP-D gene was transferred into the rat knee joints. (A) Rats were injected intra-articularly with rAAV encoding SP-D-specific shRNA for 10 consecutive days and sacrificed at the fourth week of the first injection. H&E staining and immunohistochemical staining of SP-D, CD68, F4/80, and TLR4 protein levels in cartilage were assessed following rAAV-GFP or rAAV-SP-D shRNA injection into the rat knee joints. The ratios of immunoreactive cells of SP-D (B), CD68 (C), F4/80 (D), and TLR4 (E) were analyzed. Data were expressed as mean ± SEM (n = 5). **P < 0.01 and ***P < 0.001 vs. rAAV-GFP group.

Fig 3: SP-D modulated TLR4-mediated Akt signaling in vivo. (A) Immunofluorescence with an antibody to p-Akt in articular cartilage from the ACLT + MMx -induced OA rats with rhSP-D and TAK-242 treatment at 10 weeks post-surgery. (B) The ratios of immunoreactive cells were quantified in articular cartilage according to immunofluorescence. Data were expressed as mean ± SEM (n = 5). ***P < 0.001 vs. the sham-operated group; ###P < 0.001 vs. the OA-induction group; &&&P < 0.001 vs. OA + rhSP-D group; @@@P < 0.001 vs. OA + TAK-242 group.

Fig 4: Expression of SP-A and SP-D in lung tissue of mice. (A and C) Representative immunohistochemical images showed reduced SP-A and SP-D expression in the SM group (400x). The black arrow points to the positive expression of pulmonary surfactant protein staining. Scale bar: 20 µm. (B and D) Ratio of SP-A or SP-D positive pneumocytes II to total II pneumocytes decreased in SM group. (E and F) Western blot analysis of lung tissue lysates showed that the relative expression of SP-A and SP-D decreased in COPD model. ***P<0.001.

Fig 5: An overview of study timelines and suppression of inflammatory and immune responses by SP-D in the rat OA model. (A) The ACLT + MMx rats were put into an electronic rotator cage for 30 min per day as a means of inducing OA model beginning 1 week post-surgery. At 4 weeks post-surgery, animals were injected intra-articularly with different concentrations of rhSP-D and TAK-242 once per week. PBS was used as controls in sham and OA model animals. At 10 weeks post-operation, the animals were euthanized by cardiac exsanguination. Histological staining, immunohistochemistry, immunofluorescence, and TEM were used for detection. (B) Microscopic observation and synovial immune cells infiltration were assessed via TEM (3,000 ×). 1 = macrophage;2 = neutrophil;3 = lymphocyte;4 = synovial fibroblast;The red arrows represented lysosomes. (C) Immunohistochemical staining of TLR4 and TNF-a in ACLT + MMx-induced OA rats with the administration of rhSP-D and TAK-242. The ratios of immunoreactive cells were quantified. Data were expressed as mean ± SEM (n = 5). ***P < 0.001 vs. the sham-operated group; #P < 0.05 and ##P < 0.01 vs. the OA-induction group; &P < 0.05 vs. OA + rhSP-D group; @P < 0.05 and @@P < 0.01 vs. OA + TAK-242 group.