Effect Of Xuebijing Injection On The Lung And Kidney Tissues And Its Protection Mechanism In Septic Rats

Objective: Sepsis is defined as a systemic inflammatory response syndrome which was originated by infection. Up to date, many studys indicated that: the mortality rate of sepsis and its main complication, multiple organ dysfunction syndrome( is MODS), is more than 70%. Endothelin-1 (ET-1), a peptide of 21 amino acid residues, is the most potent vasoconstrictor substance as known. In pathological situation, the concentration of ET-1 in plasma develops high, and the binding of ET-1 to its cell surface coupled receptor induces constriction of vascular beds and hypoperfusion, which results in a high incidence of multiple organ dysfunction syndrome or multiple organ failure. In sepsis, endotoxin can induce many kinds of cells to express inducible nitric oxide synthase (iNOS) and then release a lot of nitrogen monoxide (NO), which results in tissue's injury. Recent evidence suggests that degradation of the epithelial extracellular matrix (ECM) by specific enzymes known as matrix metalloproteinases (MMPs) may contribute to the development of tissue injury. A subgroup of these enzymes known as collagenases including MMP-9 (also known as gelatinase B) can degrade basement membrane components including typeⅣcollagen, fibronectins, and gelatin. Increase in MMP-9 activity leads to increased degradation of the ECM during tissue injury. Tissue inhibitors of metalloproteinases (TIMPs) is the specific physiological inhibitor of MMPs. And TIMP-1 can inhibit MMP-9 specificly.In this study, in order to elucidate the protective effects of Xuebijing injection on sepsis and explore a new method to treat sepsis and MODS, we duplicated the model of sepsis by cecal ligation and puncture (CLP), and then the effects of Xuebijing injection on expression of ET-1,iNOSmRNA, MMP-9,TIMP-1mRNA in lung and renal tissues in septic rats were systematically investigated. Meanwhile histomorphological and ultrastructural changes of tissues were also observed .Method: The method of cecal ligation and puncture (CLP) was used to duplicate severe sepsis model. Thirty healthy male Sprague-Dawley rats (250-300g) were randomly divided into 3 groups:①Sham group(n=5): in which the rats'abdomen just was incised and then sutured;②CLP Model Group(NS group n=10): administered with saline (4mL/kg) through vena caudalis injection after CLP;③Xuebijing Group(n=15): in which rats were treated with Xuebijing (4mL/kg) through caudalis injection after CLP.At 6h after CLP, the rats were sacrificed, Their lower lobes of right lung and right kidneys were resected and histopathological characteristic was observed by light microscopy and transmission electron microscopy technique. And their middle lung lobes of right lung were also resected from which excess fluid was blotted from specimens. Wet weight was measured immediately and dry weight was measured after drying specimens at 85°C for 24h to constant weight. And then the lung wet/dry weight ratio was then calculated. Meanwhile their left kidneys were resected for detection of ET-1,iNOSmRNA, MMP-9,TIMP-1mRNA with reverse transcription-polymerase chain raction (RT-PCR) and were semi-quantitatively analyzed.Statistical analysis was performed using SPSS 13.0 software package. Data were expressed as mean±SEM( x±s), and the means of each group were analysed with one-way ANOVA. A level of P<0.05 was considered statistically to be significant.Results:1 The general situation of rats in this experiment was abserved: The rats in the Sham group analepsied earlier than those in other groups and can act more actively. No abdominal dropsy and evil smelling were found when their abdomen were opened. The rats in NS group analepsiaed later, and depressed with inaction, piloerection, and tachypnea. When the abdomen was opened, bloody ascites and evil smelling was found and bowels looked edema. Cecum was darkened in some rats. Compared with NS group, the manifestations in the rats of xuebijing injection group were better.2 The expressions of ET-1mRNA,iNOSmRNA and MMP-9 mRNA in lung tissue and renal tissue in NS group were significantly increased compared with that in Xuebijing group and Sham group. However there was no difference between NS group and Xuebijing group in TIMP-1mRNA expression.3 Change of the lung wet/dry (W/D) weight ratio in septic rats showed that the lung W/D was significantly increased in NS group at 6h after CLP (P<0.01 vs Sham group: 5.15±0.11 vs 4.54±0.16); The lung W/D was significantly decreased in X group at 6h after CLP (P<0.01 vs NS group: 4.74±0.08 vs 5.15±0.11).4 The histopathological changes of lung tissue were observed. Light microscopy showed: In sham group, the lung structure including alveolar space and alveolar septum was normal; In NS group, the lung was heavy and swelling in dark red color with diffuse spots. The lung parenchyma was curmbly and rich in erythroic edema fluid. There was alveolar septum widening, capillary congestion, interstitial and alveolar edema, intra-alveolar hemorrhage and multiple inflammatory cell infiltration in the lungs of the NS group. The histopathological changes in Xuebijing group were not as serious as that in NS group. Electron microscope showed that the microvilli and organella were nomal in Sham group, but the lungⅡtype cell chondriosome disappeared and the lamellar body was evacuated in NS group. There were fewer changes in the Xuebijing group, either.5 The histopathological changes in renal tissue were observed by light microscope. In sham group, the structures of renal glomerulus and tubuli were normal, A few inflammatory cells were found in renal interstitium. In NS group, renal glomerulus shrinked obviously.Meanwhile,renal proximal tubular epithelial cell was degenerated, necrosised and shed. In addition, dilatation of renal capsular space and a lot of mononuclear leukocytic infiltration in renal interstitium were found. Compared with NS group, the changes of renal glomerulus and renal tubuli in Xuebijing group were better.The ultrastructural changes in renal tissue were observed by electron microscope: In Sham group, the tissue was almost normal. In NS group, glomerular basement membrane was thicken and humped, and some foot process cells conjugated. Furthermore, some vascular endothelia cells hyperplasied and became thicken. In liberation face of proximal convoluted tubule, the quantity of microvillus was decreased and arranged irregularly. Some crista mitochondriales were found and mitochondria membrane fusion decreased or disappeared. Rough endoplasmic reticulum stretched lightly, and the phenomenon of degranulation was obvious. Compared with NS group, ultrastructure changes of renal glomerulus and proximal convoluted tubule in Xuebijing group were better.Conclusions:1 The sepsis model could be made by cecal ligation and puncture (CLP) successfully.2 The severity of sepsis and MODS was related with the levels of ET-1,iNOSmRNA and MMP-9,TIMP-1mRNA intimate- ly, the higher concentration of ET-1,iNOS and MMP-9, TIMP-1, the more damage to tissue functions.3 Xuebijing could protect lung and renal function in septic rats, which may be related with the level decrease in ET-1,iNOS and MMP-9mRNA.