|
Objective To establish a mouse endotoxin (lipopolysaccharide, LPS) acute kidney injury (ALI) model, dynamic to observe sophoridine the treatment of endotoxin-induced renal injury in mice model, research Sophoridine LPS signal transduction pathways recognition receptor LBP, CD14, TLR4, intracellular NF-kappaB pathway IKK, IkappaB signal molecules, nuclear factor kappa B and inflammatory mediators TNF-alpha impact to investigate the pharmacological mechanism of the anti-endotoxin. Methods BALB / c mice were randomly divided into six groups, normal control group, endotoxin renal injury in LPS group, the treatment group sophoridine sophoridine high (12mg/kg), in (6mg/kg), low (3mg / kg) three dose sophoridine the the drugs control group (12mg/kg). Each group of mice were further divided into 2h, 6h, 12h, 24h four time points. To intravenous injection of endotoxin 7mg/kg preparation of acute kidney injury in a mouse model. Mice in each group, respectively, in the corresponding time points after treatment is completed, ie, 2h, 6h, 12h, 24h when the to record general condition and drawn, the naked eye and microscopic observation of renal pathological changes; urine enzymatic determination of serum urea nitrogen (BUN) content; radioimmunoassay of serum tumor necrosis factor (TNF-alpha) content; renal tissue was detected by reverse transcriptase polymerase chain reaction (RT-PCR) LBP, CD14, TLR4, IKK-beta, IkappaB-beta, p65 and TNF -alpha mRNA expression levels; immunoblotting (Western-blot) detection of P65 protein expression in renal tissue. Immunohistochemical SABC renal tissue was detected in the phosphorylation of IKK and p65 protein levels. LPS group mice (1) toxins kidney injury after administration of LPS 30 minutes of the onset of symptoms, no relief to 24h; naked eye and light microscopy to observe the pathological changes found 6h from kidney pathological changes visible by light microscopy, 12h visually observable lesions, and gradually increased to 24 are the most serious; beginning to show Sustained increases in serum BUN values ??from 2h to 24h yet to see signs of decline (P lt; 0.05) The serum TNF-α2h in began to increase and reached a peak of 6 hours and 12h began a slow decline, but still the same point in time up to 24h normal control group was significantly higher (P lt; 0.01). LPS renal tissue LBPmRNA, CD14mRNA with TLR4mRNA expression began to increase at 2h, 6h or 12h and reached the peak at 24h remained high (P lt; 0.05) The LPS group renal tissue IKK-beta, IkappaB-beta mRNA peaked 2h point in time, then begin to decline, 24h time point is still significantly higher than normal control group. NF-kappaB p65 mRNA and protein in LPS group was significantly higher respectively peaked at 6h and 12h to 24h time point decline in the same point in time but still higher than the normal control group (P lt; 0.01) Immunohistochemical detection of immunohistochemistry IOD values ??to detect the LPS group P65 and phosphorylation of IKK protein expression was sustained rise. (2) sophoridine three doses varying degrees to improve the general condition of the endotoxin-induced renal injury in mice, reduce the damage of renal pathological; sophoridine the changing trends of the alkali three-dose treatment group BUN value similar to the LPS group were beginning to show self-2h continues to rise, but with the LPS group 12h after the three-dose treatment group BUN values ??are rapidly declining, to 24 have been reduced to normal levels. Sophoridine alkali, high-dose mice treated with serum TNF-alpha were significantly lower than the same point in time LPS group (P lt; 0.01 or P LT; 0.05) Compared with LPS group, sophoridine three-dose treatment group were significantly varying degrees down to the same point in time LBP, CD14, TLR4 mRNA expression. Compared with LPS group, sophoridine alkali three-dose treatment group were significantly down-regulated to varying degrees at the same time point IKK-beta, IkappaB-beta, and NF-kappaB P65 mRNA expression. IOD value of the immunohistochemical detection of phosphorylation of IKK protein expression of display Huai fixed alkali, low-dose treatment group in 6h, 12h, 24h time point were significantly lowered the phosphorylation of IKK protein expression; and 24h time point of the middle dose group phosphate of IKK protein expression in normal control group with the same point in time, there was no significant difference (P gt; 0.05). Western-blot detection display sophoridine treatment in dose group at each time point P65 protein expression was significantly reduced (P lt; 0.05) compared with the same time point LPS group, and 24h time point has been with the same point in time normal control group, no significant difference . While high, the low-dose group at some point in time the performance of a significant downward effect (P lt; 0.05). Immunohistochemical detection of the sophoridine treatment group and LPS group compared to the same time point p65 cytoplasmic expression and nuclear translocation were weakened. Conclusions (1) observed LPS-induced endotoxemia acute kidney injury in mice 2h, 6h, 12h and 24h the general condition of renal pathological damage, serum BUN, TNF-alpha content kidney organizations LBP, CD14, TLR4 , IKK, IkappaB-beta, NF-kappaB P65 and TNF-alpha mRNA expression levels, renal tissue NF-kappa B P65 and phosphorylation of IKK protein expression trends. (2) sophoridine (12mg/kg), (6mg/kg), low (3mg/kg) three doses intervention endotoxemia in mice with acute kidney injury has a protective effect. (3) sophoridine anti-endotoxin kidney injury mechanism and inhibition downstream of inflammatory cytokines TNF-alpha release, and its role may be down model of mouse renal tissue LPS recognition receptors LBP, CD14, TLR4 expression, down NF- kappaB pathway of signal transduction molecule IKK-beta, IkappaB-beta, NF-kappaB p65 expression, inhibition of the phosphorylation of IKK in kidney tissue to interfere with NF-kappaB gene transcription. (4), high-dose Huai given alkali lowered TNF-alpha expression of significantly stronger at low doses, with a significant amount - effect relationship; while sophoridine alkali three dose groups the other observations indicators of role there was no significant the amount of - effect relationship. (5) In the present study sophoridine all test data There were no significant differences between the alkali drug control group and normal control group, confirmed sophoridine (12mg/kg) of LPS recognition receptors in normal mice and NF-kappaB pathway no significant effect.
|