| Intestinal ischemia-reperfusion (I-R) injury is a classical model reflecting acute stress and inflammatory responses, during which process the body suffers severe metabolic disorders and out-of-control inflammatory responses. Leptin is a bio-active protein secreted specifically by adipose tissue, which can inhibit food intake and promote energy expenditure; while orexin-A is another bio-active protein secreted by specific neurons in lateral hypothalamus, mainly promote food intake and suppress energy expenditure. As those two factors correlate closely with energy metabolism and former researches have disclosed the adverse change of their levels in metabolic conditions, we hypothesized that their levels might undergo a certain degree of fluctuation in the severe metabolic disorders of intestinal I-R injury. We established a rat intestinal I-R injury model, detected the proteic and mRNA expression levels of these two factors, and tried to find out the potential role they take as inflammatory cytokines. Our work mainly consisted of the following three parts:1. Establishment of intestinal I-R injury model and collection of samples(1) Rats were divided randomly into six groups: Sham, 60 min-ischemia/30 min-reperfusion (I60'R30'), I60'R90', I60'R150', I60'R240' and I60'R360'; each group contained nine rats. After anesthesia and sterilization, we opened rats' abdominal cavity and separated superior mesenteric artery. Clipped it for 60 min with arteriole clips, then released for reperfusion.(2) Drew blood from tail vein before I-R injury, while from heart after injury. Separated serum and plasma for detecting leptin and orexin-A, respectively. After blood withdrawal, cut out epididymal fat pad on the leftside and separated hypothalamus tissue, snap-frozen in liquid nitrogen and stored in -80 C for later extraction of proteic leptin and orexin-A or their total RNA.2. Effect of intestinal I-R injury on leptin levels in serum and adipose tissue(1) We used a murine leptin radioimmunoassay established firstly in our country by ourselves and an adjusted method to detect serum and adipose leptin concentrations. Results showed that: Compared with before injury, serum leptin level of I60'R30' decreased significantly, that of I60'R150' expressed a trend to increase, and that of I60'R360' increased significantly. Compared with sham group after injuy, serum leptin level of I60'R360' increased significantly, while that of I60'R240' expressed a trend to increase; adipose leptin levels of I60'R30' and I60'R90' decreased significantly, and that of 160'R3 60' increased significantly.(2) We used Western Blot to detect adipose leptin level, but results showed that: As the comparatively lower level of leptin in adipose tissue, there was insufficient sensitivity of SDS-PAGE and Western Blot to detect leptin levels in adipose tissue; moreover, there were some strong non-specific luminescence belts.(3) We used RT-PCR to detect leptin mRNA expression in adipose tissue, results showed that: Compared with sham group after injury, leptin mRNA levels of I60'R30', I60'R240' and I60'R360' increased significantly, especially I60'R30' group, while that of I60'R150' decreased significantly. Interestingly, the PCR product size was about 290 bp, quite different from the size provided by the reference paper.3. Effect of intestinal I-R injury on orexin-A levels in plasma and hypothalamus(1) We established a human orexin-A radioimmunoassay firstly in ourcountry, and used it to detect rat plasma and hypothalamus orexin-A levels, as human orexin-A is homogenous with rat orexin-A. Results showed that: Compared with before injury, plasma orexin-A levels of each group showed no significant difference. Compared with sham group after injury, plasma orexin-A levels of any other group showed no significant difference, as well as hypothalamus orexin-A levels of any other group showed no significant difference.(2) We used RT-PCR to detect orexin-A mRNA expression in hypothalamus tissue,...
|
PDF Full Text Request