CCK-8 inhibits expression of TNF-alpha in the spleen of endotoxic shock rats and signal transduction mechanism of p38 MAPK

Abstract

Aim: To study the effect of sulfated cholecystokinin-octapeptide (CCK-8) on systemic hypotension, gene and protein expression of TNF-alpha in the spleen of lipopolysaccharide (LPS)-induced endotoxic shock (ES) rats, and further investigate the signal transduction mechanism of p38 mitogen-activated protein kinase (MAPK).

Methods: The changes of blood pressure were observed using physiological record instrument in the four groups of rats: LPS (8 mg x kg(-1), iv), CCK-8 (40 microg x kg(-1), iv) pretreatment 10 min before LPS (8 mg x kg(-1)), CCK-8 (40 microg x kg(-1), iv) or normal saline (control) group. The content of TNF-alpha in the spleen was assayed 2 h after LPS administration using ELISA kit and the expression of TNF-alpha mRNA was examined 30 min, 2 h and 6 h after LPS administration by reverse transcribed polymerase chain reaction (RT-PCR). Activation of p38 MAPK was detected with Western blot 30 min after LPS administration.

Results: CCK-8 reversed LPS-induced decrease of mean arterial pressure (MAP) in rats. The content of TNF-alpha in the spleen was (282+/-30) ng x L(-1) in control group, while it increased to (941+/-149) ng x L(-1) in LPS group, P<0.01. CCK-8 significantly inhibited the LPS-induced increase of TNF-alpha content in spleen. It decreased to (462 +/-87) ng x L(-1) in CCK-8+LPS group, P<0.01. The expression of TNF-alpha mRNA 30 min and 2 h after treatment was stronger in LPS group, while it was lowered after CCK-8 pretreatment. The p38 MAPK expression increased significantly in LPS group (5.84 times of control) and CCK-8 increased the activation of p38 MAPK in ES rats (10.74 times of control).

Conclusion: CCK-8 reverses the decrease of MAP in ES rats and has inhibitory effect on the gene and protein expression of TNF-alpha in spleen, and p38 MAPK may be involved in its signal transduction mechanisms.

Figure 1

Mean arterial pressure (MAP) of animals injected normal saline, LPS, CCK + LPS and CCK, n = 6. ^aP < 0.05, ^bP < 0.01, vs Control, ^cP < 0.05, ^dP < 0.01, vs LPS

Figure 2

Effects of CCK on TNF-α , IL-1β and IL-6 2 h(TNF-α) or 6 h (IL-1β and IL-6) following LPS administration. n = 6. ^aP < 0.05, ^bP < 0.01, vs Control; ^cP < 0.05, ^dP < 0.01, vs LPS.

Figure 3

Effect of CCK-8 on LPS-induced TNF-α mRNA expression. Total RNA from the rat spleen was extrated at 30 min(A) or 2h(B) after LPS administration. n = 3. ^aP < 0.01 vs Control; ^bP < 0.01 vs LPS.

Figure 4

CCK-8 increases p38 MAPK activationon induced by LPSin spleen. ^aP < 0.05, ^bP < 0.01 vs Control; ^cP < 0.01 vs LPS.