Cobra Venom Factor-induced complement depletion protects against lung ischemia reperfusion injury through alleviating blood-air barrier damage

Abstract

The purpose of this study was to study whether complement depletion induced by pretreatment with Cobra Venom Factor (CVF) could protect against lung ischemia reperfusion injury (LIRI) in a rat model and explore its molecular mechanisms. Adult Sprague-Dawley rats were randomly assigned to five groups (n = 6): Control group, Sham-operated group, I/R group, CVF group, I/R + CVF group. CVF (50 μg/kg) was injected through the tail vein 24 h before anesthesia. Lung ischemia reperfusion (I/R) was induced by clamping the left hilus pulmonis for 60 minutes followed by 4 hours of reperfusion. Measurement of complement activity, pathohistological lung injury score, inflammatory mediators, pulmonary permeability, pulmonary edema, integrity of tight junction and blood-air barrier were performed. The results showed that pretreatment with CVF significantly reduced complement activity in plasma and BALF. Evaluation in histomorphology showed that complement depletion induced by CVF significantly alleviated the damage of lung tissues and inhibited inflammatory response in lung tissues and BALF. Furthermore, CVF pretreatment had the function of ameliorating pulmonary permeability and preserving integrity of tight junctions in IR condition. In conclusion, our results indicated that complement depletion induced by CVF could inhibit I/R-induced inflammatory response and alleviate lung I/R injury. The mechanisms of its protective effects might be ameliorated blood-air barrier damage.

Figure 1

CVF decreases complement activity in plasma and BALF. (A) Graphic presentation of CH50 in serum. (B) Graphic presentation of C3 in plasma. (C) Graphic presentation of C3 in BALF. (^*P < 0.05, compared with control. ^#P < 0.05, compared between I/R and I/R + CVF group).

Figure 2

Morphological changes in Control, Sham, CVF, I/R and I/R + CVF groups by H&E staining(×200) (A). The scoring of lung injury shown in (B). (^*P < 0.05, compared with control. ^#P < 0.05, compared between I/R and I/R + CVF group).

Figure 3

CVF pretreatment inhibits I/R-induced IL-1β, IL-6, TNF-α and MPO in lung tissues and BALF. (A) Graphic presentation of IL-1β abundance in lung tissues. (B) Graphic presentation of IL-1β abundance in BALF. (C) Graphic presentation of IL-6 abundance in lung tissues. (D) Graphic presentation of IL-6 abundance in BALF. (E) Graphic presentation of TNF-α abundance in lung tissues. (F) Graphic presentation of TNF-α abundance in BALF. (G) Graphic presentation of MPO activity in lung tissues. (H) Graphic presentation of MPO activity in BALF. (^*P < 0.05, compared with control. ^#P < 0.05, compared between I/R and I/R + CVF group).

Figure 4

CVF pretreatment decreases I/R-induced pulmonary permeability and edema. (A) Graphic presentation of the Wet/dry ratios in lung tissues. (B) Graphic presentation of total proteins in BALF. (C) Representative Western blot images of AQP1 in lung tissues. (D) Graphic presentation of AQP1 abundance in lung tissues (The grouping of gels/blots cropped from different parts of the same gel). (^*P < 0.05, compared with control. ^#P < 0.05, compared between I/R and I/R + CVF group).

Figure 5

CVF pretreatment preserves integrity of tight junctions and blood-air barrier in IR condition. (A) Representative Western blot images of ICAM1 in lung tissues (The grouping of gels/blots cropped from different parts of the same gel). (B) Graphic presentation of ICAM1 abundance in lung tissues. (C) Representative Western blot images of ZO-1 in lung tissues (The grouping of gels/blots cropped from different parts of the same gel). (D) Graphic presentation of ZO-1 abundance in lung tissues. (E) Representative Western blot images of MMP2 in lung tissues (The grouping of gels/blots cropped from different parts of the same gel). (F) Graphic presentation of MMP2 abundance in lung tissues. (G) Representative Western blot images of MMP9 in lung tissues (The grouping of gels/blots cropped from different parts of the same gel). (H) Graphic presentation of MMP9 abundance in lung tissues. (^*P < 0.05, compared with control. ^#P < 0.05, compared between I/R and I/R + CVF group).