Transient limb ischemia alters serum protein expression in healthy volunteers: complement C3 and vitronectin may be involved in organ protection induced by remote ischemic preconditioning

Ting Pang, Yang Zhao, Nan-Rong Zhang, San-Qing Jin, San-Qiang Pan, Ting Pang, Yang Zhao, Nan-Rong Zhang, San-Qing Jin, San-Qiang Pan

Abstract

The protective mechanism underlying remote ischemic preconditioning (RIPC) is unclear. This study aims to verify whether the protein expression profile in the serum could be altered by RIPC and to detect potential protein mediators. Transient limb ischemia consisting of three cycles of 5-min ischemia followed by 5-min reperfusion was performed on sixty healthy volunteers. Serum samples were collected at 30 min before transient limb ischemia and at 1 hour (h), 3 h, 8 h, 24 h, and 48 h after completion of three cycles. Changes in the serum protein profile were analyzed by two-dimensional gel electrophoresis and proteins were identified by MALDI-TOF/TOF mass spectrometry. Fourteen differentially expressed proteins were identified and, respectively, involved in immune system, lipid binding and metabolism, apoptosis, and blood coagulation. Complement C3, vitronectin, and apolipoprotein A-I were further confirmed by western blotting, and the results showed that their contents decreased significantly after transient limb ischemia. It is concluded that transient limb ischemia alters the serum protein expression profile in human being, and that reduction of serum contents of complement C3 and vitronectin may represent an important part of the mechanism whereby RIPC confers its protection.

Figures

Figure 1
Figure 1
Representative images of SYPRO-Ruby-stained 2-DE gels. Representative images of SYPRO-Ruby-stained 2-DE gels at various time points ((a) before transient limb ischemia, (b) 1 h after transient limb ischemia, (c) 3 h after transient limb ischemia, (d) 8 h after transient limb ischemia, (e) 24 h after transient limb ischemia, and (f) 48 h after transient limb ischemia). The high-abundant proteins such as albumin and immunoglobulins were depleted from serum using the multiple-affinity column, as described in Section 2. Zoomed areas highlight typical spots (arrows) of the fourteen differentially expressed proteins. Changes in these spots' intensity among different time points are clearly visible. The spot numbers refer to proteins summarized in Table 2.
Figure 2
Figure 2
Peptide mass fingerprinting spectrum and a typical MS/MS map of complement C3. (a) Peptide mass fingerprinting spectrum of complement C3. The arrow indicates the peptide detected at m/z 1083.5814. (b) A typical MS/MS map of complement C3. The sequence of precursor at m/z 1083.5814 (arrow in (a)) was analyzed in this map.
Figure 3
Figure 3
Western blotting data of complement C3, vitronectin, and apoA-I. The results were presented as mean ± SD. Error bars were SD. Repeated-measures analysis of variances was performed to evaluate statistical significance.*P < 0.05. (a) Representative western blotting images of serum samples from healthy volunteers obtained prior to transient limb ischemia (base) and at various time points (1 h, 3 h, 8 h, 24 h, and 48 h) thereafter. Memcode was shown to demonstrate equal protein loading. (b) Changes in the expression of complement C3 after transient limb ischemia compared with that before transient limb ischemia (n = 60). (c) Changes in the expression of vitronectin at 75 kDa after transient limb ischemia compared with that before transient limb ischemia (n = 60). (d) Changes in the expression of vitronectin at 65 kDa after transient limb ischemia compared with that before transient limb ischemia (n = 60). (e) Changes in the expression of apoA-I after transient limb ischemia compared with that before transient limb ischemia (n = 60).

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