| Objective:Sepsis was initiated by the infection of systemic inflammatory response syndrome(SIRS), and extremely easy to develop into the septic shock and the multiple organ dysfunction syndrome (MODS), as one of main causes to death of the clinical critically ill patients, its mortality was especially higher when combined with acute renal failure (ARF). But there was still a lack of clinical effective prevention measures in view of the bacterial sepsis at present,the treatment of sepsis has still been one difficult problem ,which puzzled the medical workers. The inoculation fluid of xuebijing has the function to improve blood circulation, remove the extravasated, improve vascular permeability, reduce the inflammation reaction. This research plans to establish the mouse-model with the caecum ligation perforation operation (CLP) through the method of CLP,which was highly related to the clinical sepsis,by observing the renal pathological changes and the expression of vascular endothelial growth factor(VEGF) in the renal tissue, to monitor the changes of blood serum tumor necrosis factor alpha(TNF-α),interleukin-6(IL-6),further explored the pathophysiologic mechanism of renal injury with sepsis and the protective function of the treatment with xuebijing, to provides the new thinking for the clinical prevention and treatment of sepsis.Methods:The method of CLP was used to duplicate severe sepsis model.One hundred and forty-four healthy adult Spragne-Dawley(SD) rats(200～230g)were randomly divided into four groups: The normal control group (group A,n=24),sham-operation group (group B,n=24), sepsis-model group (group C,n=48) and treatment group with xuebijing(group D,n=48). Each group divided into four sub-groups, according to time sequence:6h, 24h, 48h, 72h after the CLP operation,group A and group B contained 6 rats, the later two groups contained 12 rats. Each group of rats were anaesthetized with 10% chloral hydrates(3ml/kg) before the operation.And the treatment group with xuebijing were gived the inoculation fluid by the intravenous injection,respectively in 0h, 12h, 24h, 36h, 48h,60h after the operation,each time the dosage was 4ml/kg;Sepsis-model group were parallelly injected the same dose of saline in the same way. All groups gived the hypodermic injection with 10ml 0.9%Nacl for anti-shock treatment after the CLP operation.It started to record the time when the CLP operation finished,after giving animal anaesthesia in the corresponding time 6h, 24h, 48h, 72h, picked the blood via the abdomen aorta, useing the enzyme-linked immunosorbent assay(ELISA) method to determine the density of blood serum concentration of TNF-αand IL-6,simultaneously monitored the level of blood serum urea nitrogen (BUN) and creatine(Cr) in the same time.Picked up the left renal,which was fixed by 4% paraformaldehyde, maded into the paraffin section, to observe the pathology morphological changes under the light microscope with the technique of hematoxylin-eosin(HE) dyeing, to examine the expression of VEGF with the immunity histochemistry.Results: 1 The general observation of each group: Group A:There were no deaths, the diet, the feces and urine were normal, respond keenly,breath steadily,the wool was smooth and glossy, like to huddle together for warming up.Group B was completely sober in 30min more or less after operation, the general situation was same with group A,when cut up the belly,without performance of evil smelling and dropsy necrosis intestines. Group C of rats appeared the consciousness detention,the energetic dispirited after awaking, the body crouching almost no activity,a small amount of water ,unresponsive to the environment , respiratory rate faster,the wool was fluffy and less glossy,the feces was thin and soft,no longer crowding together for warming .Rats were dead within 24h.When cut up the belly can be seen the wicked stink courage and uprightness to infiltrate the fluid obviously, the intestines were dropsy and adhered, the caecum was of necrosis and nigrescence. Group D compared with group C, the clinical manifestation reduced obviously,and the number of death cases reduced.2 each group of rats renal histology changes: Group A:the structure of renal tissue was integrated,no edema, vacuolar denaturation and necrosis. There was no obvious difference between group B and group A. Group C compared with the control group,the clarity of its tissue structure desend, a large number of inflammatory cell infiltration in renal tissue, wide range of micro-thrombus in renal vascular,the renal glomerulus capillary with extravasated blood. The tubulus renalis epithelial cell vacuole changes, the arrangement disorder, the acorn tube lumen was narrow and small, mesenchymal hyperemia and dropsy. Even with the karyopyknosis, cell structure vanishing.The pathology damages of Group D lightly compared with group C, and the tendency of the reduction is obvious.3 Biochemistry target observation:In this experiment all biochemistry target group A did not have statistics significance with group B(P<0.05).The level of blood serum TNF-αelevated obviously in 6h (42.37±2.96pg/ml) after CLP operation in group C, and had maintained the higher level;The level of IL-6 also obviously elevates in 6h (197.96±6.23pg/ml), achieved the peak (520.00±29.15pg/ml)in 24h; The level of BUN started to elevate obviously (14.90±0.71mmol/L) from 6h, the level of Cr elevated later than BUN, obviously elevated(106.73±7.45μmol/L) in 24h, compared with group A and group B,the difference had statistics significance (P<0.05). The level of TNF-α, IL-6, BUN, Cr was obviously lower than no treatment group in the same time (P<0.05) in group D.4 The VEGF expression of renal tissue: The main coloration spot of VEGF was the cytoblastema. Group A, group B did not express or express less, the Immunohistochemistry score(IHS) of group C obviously to elevate (1.50±0.55) in 6h, achieved the peak (9.33±2.34)in 48h, and had maintained the higher expression level, compared with group A and group B,the difference had statistics significance (P<0.05). Group D also has the expression (2.33±0.52) of 24h, compared with group C,it obviously desend, the difference had statistics significance (P<0.05),In 48h,72h there were also significant differences(P<0.05).Conclusions: 1 It was a reliable method to establish the animal renal injury model with Sepsis through the CLP operation, which was worth promoting in the experimental study related sepsis.2 TNF-α, IL-6 and VEGF were obviously elevated In the body of rats with sepsis,it explained that they possibly participated in the morbidity process renal injury with sepsis, but the concrete mechanism awaited further research.3 The targets of each group were distinctly improved after the treatment with xuebijing, it provided an experimental theory for the xuebijing applyed in the treatment of sepsis. |
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