Dynamic changes of mouse cardiac functions in early stage of sepsis induced by CLP

AIM: To detect the changes of cardiac functions of septic mice in the early stage of sepsis. METHODS: Health male Kunming mice were used in the study. The techniques of 2D, M-mode and Doppler echocardiography were applied to evaluate the cardiac functions before cecal ligation and puncture(CLP) as baseline and at time points of 12 h, 24 h, 36 h, 48 h and 168 h after CLP. The mice survived for 168 h(7 d) were considered as survivals. RESULTS: Compared to the baseline at the time point of 24 h after CLP, the blood volumes of heart return decreased significantly in the early stage of sepsis induced by CLP. LVEDV reduced by 46%. Notable compensatory responses of the hearts in septic mice were observed, especially the systolic functions, in which LVEF and LVFS increased by 27% and 39%, respectively. However, the compensatory responses of diastolic function were weaker than the systoles. E/A ratio and EDT decreased by 30% and 25% respectively at the time point of 24 h. CONCLUSION: The strong compensatory cardiac functions are one of the factors for supporting the septic animal to survive. Protection of the cardiac functions especially the diastoles is important in the treatment of septic patients.     

The protective effect of cistanche deserticola Y.C Ma. on thymocytes in septic rats

AIM: To study the protective effect of cistanche deserticola Y. C Ma. on thymocytes in septic rats. METHODS: Cecum ligation perforation (CLP) was used to induce sepsis. Treatment group was treated with cistanche deserticola Y. C Ma. （1.25 g·kg-1·d-1， ig） for 14 days before CLP. Animals were killed 12 h or 24 h after CLP and thmocytes were collected. The ratio of thmocyte apoptosis and mitochondrial membrane potential were determined by the flow cytometry. The ATP activity was detected by spectrophotography. RESULTS: The rate of thmocyte apoptosis significantly increased 12 h after CLP. The ATP activity decreased 24 h after CLP was significant. The extract of desert living cistanche effectively repressed the apoptosis of thymocytes and maintained the mitochondrial membrane potential. CONCLUSIONS: The extract of cistanche deserticola Y. C Ma. protects thymocytes against apoptosis induced by sepsis. Maintaining of mitochondrial membrane potential may be the protective mechanism.     

Protective effect of ginkgolide B on CLP-induced septic mice

AIM: To explore the potential immunomodulatory effect and related mechanisms of ginkgolide B (GB), a known potent antagonist of platelet-activating factor receptor, on the pathological process of sepsis. METHODS: The experimental sepsis model was established by a standardized procedure of cecal ligation and puncture (CLP). GB treatment (10 μg/g) was given to the CLP mice 30 min before the surgical operation. The survival rate was observed every day for 3 weeks. The NO content in the serum was measured by Griess assay. The ROS level in the blood was determined by H₂DCFDA labeling and flow cytometry. The inflammatory cytokines in the serum were detected by the methods of cytometric bead array and ELISA. RESULTS: The thymus and spleen of the mice significantly atrophied, and the levels of NO, ROS, TNF-α, IL-1β, IL-6 and IL-10 in the blood were dramatically elevated 24 h after CLP. All the CLP mice died in 5 days. However, treatment with 10 μg/g of GB 30 min before CLP remarkably enhanced the indexes of thymus and spleen, inhibited the storm of inflammatory mediators and cytokines, and improved the survival rate. CONCLUSION: The protective effect of GB on CLP-induced experimental sepsis indicates that GB is a candidate of natural immunomodulator for treating sepsis.     

Dynamic expression of CD30 on peripheral blood lymphocytes in mice after a cecal ligation and puncture

AIM: To investigate the details of Th2 cell differentiation in septic mice. METHODS: Experimental septic mice were induced by cecal ligation and puncture (CLP). The exression of CD30 on CD4⁺T cells at different time after CLP were estimated by flow cytometry following three-color immunofluorescent staining.RESULTS: CD30 expression on CD4⁺T cell was different at each time point. The highest expression was showed at 38 h after CLP and declined later, which matched the changes in mortality of the animals. CONCLUSION: During sepsis, differentiation of Th2 cell changed with the development of sepsis and might be associated with the severity of the disease.     

Assessment of myocardial dysfunction in septic rats with conventional echocardiography and tissue Doppler imaging

AIM To compare various conventional and tissue Doppler echocardiographic indexes to assess their accuracy to detect left ventricular (LV) intrinsic systolic and diastolic dysfunction in septic rats. METHODS Twenty-two SD rats were randomly divided into 2 groups: sham group and sepsis group, with 11 rats in each group. A model of sepsis was established by cecal ligation and puncture (CLP). Serum tumor necrosis factor-α (TNF-α) level, and myocardial intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression were determined by ELISA and Western blot. The LV systolic and diastolic functions were analyzed by isolated heart Langendorff perfusion technique, conventional echocardiography and tissue Doppler imaging. RESULTS Compared with sham group, the serum TNF-α level, and LV ICAM-1 and VCAM-1 expression were increased significantly, while LV ±dp/dt_max, stroke volume and LV end-diastolic volume were markedly decreased at 6 h after CLP. Moreover, compared with sham group, mitral inflow peak E and A wave velocities as well as early-diastolic peak velocities (E' wave) and late-diastolic peak velocities (A' wave) of the mitral annulus were reduced significantly in sepsis group at 6 h after CLP. In contrast, no significant difference in LV ejection fraction and E/E' ratio between sham group and sepsis group was observed. Further investigation revealed that peak E' wave and A' wave velocities were positively correlated with -dp/dt_max (r=0.460 and r=0.520, P<0.05). CONCLUSION At the early stage of sepsis, tissue Doppler echocardiography is a useful technique to assess LV diastolic dysfunction, and peak E' and A' wave velocities may be the sensitive indexes to detect LV intrinsic diastolic dysfunction.     

Effects of brain tissue extract of hypoxia-preconditioned mice at different concentrations on tolerance of PC12 cells to hypoxia

AIM: To investigate the effect of brain tissue extract of hypoxia-preconditioned mice (HP extract) on tolerance of PC12 cells to hypoxia. METHODS: The mice model of acute repetitive hypoxia was reproduced and brain tissue extracts were prepared. HP extract was added into the cultures of PC12 cells and the final concentrations of HP extracts were 0.2, 0.8, 3.2, 6.4 or 12.8 g/L (HP group), respectively. Brain tissue extract of normal mice (N extract) at the same five concentrations were used as controls (N group). The PC12 cells were cultured in hypoxia (2% O2). After hypoxia for 24 h, 48 h or 72 h, colorimetric method (A570) of tetrazolium salt MTT (3-［4,5-dimethylthiazol-2-yl］-2,5-diphenyl tetrazolium bromid) staining was adopted to determine the cell viability and lactate dehydrogenase (LDH) release percentage assay was also conducted after 24 h, 48 h or 72 h hypoxia. Besides, apoptotic percentages at early stage (24 h hypoxia) and late stage (72 h hypoxia) were detected respectively by means of annexin V-FITC/PI double-stained flow cytometry and Hoechst 33258 stained fluorescence microscopy. RESULTS: HP extract at the concentrations lower than 6.4 g/L (including 6.4 g/L) showed protective effect on PC12 cells in early stage of hypoxia (24 h). A570 values in HP group were significantly higher than those in N group, but LDH release percentages were significantly lower than those in N group after 24 h hypoxia. With hypoxia prolonging, HP extract at high concentrations gradually lost the protective effect. At the time point of 72 h hypoxia, HP extract at concentrations higher than 6.4 g/L (including 6.4 g/L) had pro-apoptotic effect. At this time point, A570 values of HP groups at these concentrations were significantly lower than those in the corresponding N group, both LDH release percentages and apoptotic percentages were significantly higher than those in the N group. CONCLUSION: The effects of HP extract on tolerance of PC12 cells to hypoxia depend on its concentrations and on the time of treatment.     

Effect of electro-acupuncture at Zusanli point on tumor necrosis factor-α induced-multiple organ dysfunction in rats with sepsis

AIM: To investigate the effect of electro-acupuncture (EA) at Zusanli (ST36) on proinflammatory factors induced-multiple organ dysfunction in rats with sepsis. METHODS: Sixty four male Wastar rats were used to develop the sepsis model by cecal ligation and puncture (CLP). The animals were randomly divided into 4 groups (n=16 in each group): CLP+EA (CLP/EA), CLP+sham EA (CLP/SEA), vagotomy+ CLP+SEA (VA/CLP/SEA) and vagotomy+CLP+EA (VA/CLP/EA). Zusanli point (ST36) was electroacupunctured with constant voltage (2-100 Hz, 2 mA for 0.5 h) 20 min after CLP surgery. Bilateral cervical vagotomies were performed in rats in VA/CL/SEA and VA/CLP/EA groups. Twelve hours after CLP, animals were sacrificed and liver, kidney and jejunum were harvested for evaluating the contents of tumor necrosis factor-α (TNF-α), myeloperoxidase (MPO) and diamine oxidase (DAO). The rate of water content (WCR) of the organs was determined. At the same time, the plasma levels of alanine aminotransferase (ALT) and creatinine (Cr) in each group were also detected. RESULTS: The levels of ALT and Cr in plasma, as well as TNF-α, MPO and WCR in organ tissues were markedly lower, and the activity of DAO in jejunum tissue was obviously higher than that in CLP/SEA group at 12 h after CLP (all P<0.05). The levels of ALT, Cr, TNF-α, MPO and WCR in VA/CLP/SEA group and VA/CLP/EA group were significantly higher, the activity of DAO was obviously lower than that in CLP/SEA group (all P<0.05). No statistical difference in all above measurements between VA/CLP/EA group and VA/CLP/SEA group was observed (all P>0.05). CONCLUSION: The results indicate that EA at Zusanli point obviously decreases the levels of TNF-α in liver, kidney and jejunum tissues after CLP, and alleviates the tissue edema and dysfunction of those organs. Vagotomy decreases or eliminates the effects of EA, suggesting that activation of cholinergic anti-inflammatory pathway is one of the main mechanisms to induce the effects of EA at ST36 on CLP sepsis.     

Changes of aortic contractile reactivity and RAS components after tourniquet shock

AIM: To investigate the role of renin-angiotensin system(RAS) disequilibrium in hyporeactivity and injury of aorta after tourniquet shock(TS) by observing the changes of aortic contractile reactivity and RAS components after TS. METHODS: Male C57BL/6 mice(8 months old) were divided into 7 groups including control group and 6 model groups. The mice in model groups were sacrificed at reperfusion of 10 min, 1 h, 2 h, 4 h, 6 h and 12 h. The mice in control group were not subjected to tourniquet ligation. The Doppler flowmetry was used to determine the limb blood flow. The carotid artery catheter was applied to detect the blood pressure. The isolated vascular tension tester was available to measure the reactivity of the aorta. HE staining combined with transmission electron microscopy was used to evaluate the morphology of injured aortas. The protein expression of AT1 receptor, Mas receptor, ACE and ACE2 was measured by Western blot. The serum contents of Ang Ⅱ and Ang(1-7) were detected by ELISA. RESULTS: Compared with control group, the blood flow in model groups decreased gradually with the prolongation of reperfusion time. The blood pressure increased at 10 min after reperfusion, and then decreased gradually. Accordingly, vascular reaction to norepinephrine(NE) increased at 10 min and then descended. The vascular reactivity reached the lowest level at 4 h. Morphological injury score increased gradually. Vascular AT1 receptor and ACE2 proteins were reduced, while Mas receptor and ACE proteins were up-regulated compared with control group. The content of Ang Ⅱ in the serum elevated, while the content of Ang(1-7) was reduced. CONCLUSION: The mechanism of aortic reaction to NE increased temporarily in the early stage of shock and then decreased. It may be related to the morphological injury of aorta and the imbalance of RAS.     

Effect of portal vein transplantation with allogeneic adipose tissue-derived mesenchymal stem cells on liver fibrosis in SD rats

AIM: To explore the influence of adipose tissue-derived mesenchymal stem cells (ADMSCs) transplantation on hepatic fibrosis in rats. METHODS: ADMSCs from abdominal lipid tissues were extracted, cultured and passaged. The hepatic fibrosis rat model was built up and randomly divided into 3 groups: hepatic cirrhosis group (n=14); portal vein transplantation group (n=11) and caudal vein transplantation group (n=14). Computer tomography(CT) perfusion index, histological scores and microvessel density were detected and compared after transplantation of ADMSCs among the 3 groups. RESULTS: After transplantation of ADMSCs, the total hepatic blood perfusion, especially portal vein perfusion, significantly increased in portal vein transplantation group determined by CT perfusion scan (P<0.05), but slightly increased in caudal vein transplantation group. The histological scores showed significant alleviation of fibrosis evidence in portal vein transplantation group, and slightly change of adipose degeneration in caudal vein transplantation group. Microvessel density decreased significantly in portal vein transplantation group as compared to the other 2 groups (P<0.05). CONCLUSION: Transplantation of ADMSCs greatly helps the alleviation of hepatic fibrosis. Portal vein transplantation benefits more than caudal vein transplantation.     

Effects of berberine and yohimbine on splenocyte apoptosis in septic mice

AIM: To observe the effects of berberine and yohimbine on splenocyte apoptosis in septic mice and underlying mechanisms. METHODS: The mice were subjected to cecal ligature and puncture (CLP). The drugs or vehicle were given intragastrically 2 h after the surgery according to the following 5 groups: sham, CLP, CLP+berberine, CLP+yohimbine, and CLP+berberine+yohimbine. The apoptosis of splenocytes stained by TUNEL was observed under laser scanning confocal microscope 20 h after CLP. The splenic lymphocytes were isolated and observed using flow cytometry. The activities of caspase-3, caspase-8 and caspase-9 in splenic lymphocytes were detected, and the expression of Fas, Bim, Bcl-2 and Bax in the splenocytes was also determined by Western blotting. RESULTS: The TUNEL staining showed that the apoptotic rate of the splenocytes in septic mice 20 h after CLP was significantly higher than that in sham and CLP+yohimbine groups (P＜0.05). Compared with CLP group, the proportion of apoptotic cells was decreased in septic mice in CLP+berberine+yohimbine and CLP+yohimbine groups (P＜0.05). Flow cytometry analysis demonstrated the similar results in the apoptosis of splenocytes and T lymphocytes. However, only yohimbine treatment reduced the apoptosis of B lymphocytes in the spleen of sepsis-challenged mice. Compared with CLP group, caspase-9 activity was significantly reduced in CLP+berberine group (P＜0.05), the activities of caspase-3, caspase-8 and caspase-9 were all statistically reduced (P＜0.05) in CLP+yohimbine group and CLP+yohimbine+berberine group. CLP significantly increased the expression of cytosolic Fas, Bim and mitochondrial Bax in the splenocytes, and decreased Bcl-2 expression compared with sham group. Compared with CLP group, the expression of cytosolic Bim and mitochondrial Bax in CLP+berberine group were reduced (P＜0.05). Fas expression decreased only in CLP+yohimbine group (P＜0.05). Berberine combined with yohimbine reduced the expression of cytosolic Fas, Bim and mitochondrial Bax in the septic mouse splenocytes (P＜0.05).CONCLUSION: Yohimbine reduces sepsis-induced splenic lymphocyte apoptosis in mice by inhibiting Fas expression and in turn blocking both extrinsic and intrinsic apoptosis pathways. Berberine reduces Bim expression and inhibits caspase-9 activation, but not caspase-3 activation and apoptosis in the septic mouse splenocytes. Berberine combined with yohimbine reduces splenocyte apoptosis in the septic mice by inhibiting both extrinsic and intrinsic apoptotic pathways.     

PI3K/Akt signaling pathway regulates autophagy induced by acute kidney injury in septic rats

AIM：To investigate the autophagy induced by sepsis and acute kidney injury, and the regulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in this process. METHODS：The rats were subjected to cecal ligation and puncture (CLP) or sham operation. Histopathologic changes of the renal tissues were examined by HE staining. Blood urea nitrogen (BUN) and serum creatinine (SCr) were measured by chemical colorimetry. The protein expression of microtubule-associated protein light chain 3 I/II (LC3 I/II), beclin-1 and p-Akt at different time points after CLP was detected by Western blotting. In vitro, human proximal tubular epithelial cell line HK-2 were treated with LPS to induce autophagy. The protein expression of LC3 I/II and p-Akt in the HK-2 cells after LPS treatment at different time points and different concentrations was detected by Western blotting. These molecules in HK-2 cells and apoptosis of HK-2 cells treated with LPS plus PI3K inhibitor or Akt inhibitor were also detected. RESULTS：Compared with sham group, the severe changes of renal histopathological injuries in CLP groups were observed, the levels of BUN and SCr in CLP groups were significantly increased. LC3 I/II, beclin-1 and phosphorylation of Akt gradually increased after CLP. After treatment with LPS, the expression of p-Akt (308) in the HK-2 cells gradually increased in a dose- and time-dependent fashion. The expression of beclin-1 and p-Akt (472) reached a peak at 8 h or 10 mg/L LPS treatment. Treatment with PI3K or Akt inhibitor down-regulated the expression of LC3 and promoted the apoptosis of HK-2 cells. CONCLUSION：Autophagy in the kidney is induced by sepsis and acute kidney injury. PI3/Akt signaling pathway may be involved in this process.     

Mechanisms for a decrease in mortality of LPS-challenged mice by rhynchophylline

AIM: To observe effect of rhynchophylline （Rhy） on mortality and organ injury in endotoxemic mice and further investigate the mechanisms of its actions. METHODS: Male mice were randomly assigned into control, LPS, Rhy +LPS and Rhy group, and injected subcutaneously with normal saline (0.05 mL/10 g), or rhynchophylline once a day for 3 d, 1 h after subcutaneously treatment on day 3, LPS (20 mg/kg) or normal saline was injected intraperitoneally. Survival rate was recorded every 12 h for 6 d. In another experiment, 12 h after LPS injection, the left lung and intestine tissue sections were prepared for histological analysis and the right lung were used to determine the ratio of wet to dry lung tissue weight (W/D),the serum was collected to detect the concentrations of alanine aminotransferase（ALT）, aspartate aminotransferase (AST ), bloodureanitrogen (BUN) and creatinine (Cr). In addition, the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β) and interleukin-10 (IL-10) in serum at 2 h after LPS challenge were detected by enzyme-linked immunosorbent assay. The concentration of NO in serum at 8 h was detected by enzymic method. The effect of Rhy on survival rate of mice subjected to cecal ligation and puncture (CLP) was also observed. RESULTS: Mortality of mice challenged with LPS alone was higher significantly than that in control at 24 h after LPS challenge, pretreated with Rhy at a dose of 8 or 16 mg/kg increased markedly the survival rate of LPS-challenged mice. However, Rhy at a dose of 8 mg/kg significantly increased mortality of mice subjected to CLP. In the histological analysis, severe inflammation was observed both in the lung and intestine tissues in the LPS group. LPS elevated lung W/D, the levels of ALT, AST, BUN, Cr, TNF-α, IL-1β, IL-10 and NO in serum. Pretreatment with Rhy had no obvious improvement in the lung and intestine tissue injury, and no significant depression in the lung W/D and the serum levels of ALT, AST, BUN, Cr, IL-1β, IL-10 and NO, but decreased the level of TNF-α in serum significantly in LPS -treated mice. CONCLUSION: Pretreatment with Rhy reduces the mortality in endotoxemic mice, but not decrease the mortality of mice challenged with CLP, at least in part, through inhibiting the synthesis and secretion of TNF-α.     

Establishment of a mouse model of sepsis associated encephalopathy and preliminary research of cognitive dysfunction in this model

AIM: To establish the mouse model of sepsis-associated encephalopathy (SAE) and the preliminary research of cognitive dysfunction in this model. METHODS: SPF male C57BL/6J mice of 8~10 weeks old were selected. The first part of the experiment divided the mice into 4 groups randomly, namely control group, cecal ligation and puncture (CLP)1 group and CLP2 group (CLP was performed with 7 and 12 syringe needle respectively). The mice in sham operation group were only laparotomy. In the second part of the experiment, the mice were randomly divided into control group, sham operation group and CLP group. The Kaplan-Meier method was used to analyze the postoperative survival rate of the mice in the first part experiment. The neurobehavioral scores were used to evaluate the neurobehavioral changes of the mice. The Morris water maze test and the passive avoidance experiment were used to detect the changes of cognitive memory function in the mice. The pole test and the wire suspension test were used to test the motor coordination of the mice. The serum levels of prostaglandin E2 (PGE₂) were measured by ELISA. RESULTS: In the first part of the experiment, the CLP mice showed obvious symptoms such as lethargy, piloerection, chills and anorexia. The 48 h mortality in CLP1 group and CLP2 group were 20% and 30% respectively. In the 2 parts of the experiments, the neurobehavioral scores of the CLP mice were significantly lower than those in control group and the sham operation group (P<0.01). In CLP mice, the escape latency time of the Morris water maze was significantly prolonged (P<0.01), the target quadrant dwell time and the number of crossing platforms were decreased (P<0.01), the scores in the suspension experiment and the pole test were significantly reduced (P<0.01), the activity of the mice was decreased or even did not enter the darkroom in the step-through test (P<0.05). In the second part of the experiment, the serum level of PGE₂ in the mice after CLP was significantly increased (P<0.01). CONCLUSION: A stable mouse model of sepsis-associated encephalopathy is successfully established by cecal ligation and puncture with 12 syringe needle. The SAE mouse model established by this method is useful for investigating the learning and memory cognitive dysfunction.     

Changes of potassium channels in thoracic aorta of streptozotocin-induced diabetic mice in early stage

AIM:To investigate the changes of potassium channels in thoracic aorta of streptozotocin-induced diabetic mouse in the early stage of diabetes mellitus.METHODS:The effects of 60 mmol/L KCl, phenylephrine (PE), sodium nitroprusside (SNP) were measured and concentration-response curves to SNP were determined in the presence and in the absence of the inhibitors of potassium channels on the thoracic aortic rings of diabetic and age-matched control mice in vitro. RESULTS:STZ-diabetic mice showed a significant increase in the maximum contractile response and sensitivity of thoracic aorta to 60 mmol/L KCl and PE. The endothelium-independent relaxation response to SNP was increased by diabetes and were decreased significantly by pretreatment of the vessels with 1 mmol/L tetraethylammonium (TEA), 1 mmol/L 4-aminopyridine (4-AP) and 10 μmol/L glibenclamide in diabetes thoracic aorta. Only 4-AP decreased relaxation response to SNP in age-matched control mice. The -logIC₅₀ difference of TEA in thoracic aorta rings of diabetes was significantly higher than age-matched control mice.CONCLUSION:In early stage of diabetes mellitus, the opening or expression of K_Ca channels is significantly enhanced.The opening of K_ATP channels is also enhanced in this stage.     

Naringin protects human umbilical vein endothelial cells against injury induced by high glucose through PI3K/AKT/eNOS pathway

AIM: To investigate the protective effect of naringin (Nar) on the injury of human umbilical vein endothelial cells (HUVECs) induced by 33 mmol/L high glucose (HG) and to explore its possible mechanisms.METHODS: The injury model was established by treating HUVECs with HG medium for the indicated time (6, 12, 24, 48 and 72 h), and then the levels of NO, eNOS and p-eNOS were detected, respectively. The effects of Nar on high glucose-induced endothelial cell injury were observed. HUVECs were treated with Nar at concentrations of 5, 10, 25, 50 and 100 mg/L for 6 h, 12 h, 24 h, 36 h and 48 h. The levels of NO in the supernatants were measured. The effects of Nar on HG-injured HUVECs were explored by treating the cells with 10 μmol/L of LY294002, a PI3K inhibitor, or 0.5 μmol/L of AKT inhibitor Ⅳ, an AKT inhibitor, and then the levels of NO, PI3K, AKT, eNOS and their phosphorylated proteins were determined by Western blot.RESULTS: Nar at concentration of 50 mg/L significantly attenuated the injury of endothelial cells induced by high glucose (P<0.01), and the protective effects of Nar were abolished by pretreating with the inhibitor of PI3K or AKT (P<0.01).CONCLUSION: Nar protects endothelial cells against the injury induced by high glucose through PI3K/AKT/eNOS pathway.     

Berberine inhibits enterocyte apoptosis in septic mice

AIM: To observe the effects of berberine (Ber) on enterocyte apoptosis in septic mice and its possible mechanism. METHODS: Male C57BL/6 mice (8~10 weeks old) were randomly divided into sham group, cecal ligation and puncture (CLP) group, CLP+Ber group and sham+Ber group. The mice in CLP group underwent CLP ope-ration, and the mice in sham groups suffered a similar operation except the ligation and puncture. After the sham or CLP operation, the mice were administered intragastrically with distilled water or berberine (50 mg/kg) within 2 h. After 20 h, the mice were killed with excess pentobarbital sodium and the ileum tissues were removed. The histological changes of the intestine were observed and the enterocyte apoptosis was examined by determining the protein level of cleaved caspase-3. Furthermore, mitochondrial Bax, cytoplasm cytochrome C (Cyt C) and the total proteins of Bcl-2, Fas, FasL and Fas-associated protein with death domain (FADD) were examined by Western blot. The mRNA expression of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) was measured by real-time PCR. RESULTS: The extensive ileum injuries, including remarkably increased leukocytes and necrosis of intestinal villus were observed 20 h after CLP. In CLP group, the protein levels of cleaved caspase-3, cytoplasm Cyt C, as well as Fas, FasL were significantly increased, but the Bcl-2 level was decreased. Bax translocation into mitochondria was promoted. However, FADD was not changed significantly. The mRNA expression of TH and DBH was also increased sharply in CLP group. On the contrary, treatment with berberine made a considerable alleviating alteration in the ileum of the septic mice.CONCLUSION: Treatment with berberine provides protective effects on intestinal injury in septic mice by reducing enterocyte apoptosis, and its possible mechanism may be involved in the inhibition of the endogenous and exogenous apoptosis pathways.     

Reconstruction of tissue engineering blood vessel via acellular matrix

AIM: To reconstruct the tissue engineering blood vessel by using acellular matrix of porcine thoracic aorta tissue as the scaffold, and by inoculability of vascular endothelial cells (EC) from human umbilical cord vein. METHODS: The porcine thoracic aorta was treated with 1% Triton X-100 for preparing acellular vessel matrix, and the mechanical characterization was in succession modified by freeze-drying and thermal cross-linking techniques. Meanwhile, the mechanics capability of the vessel was measured. The endothelial cells isolated from umbilical cord vein were seeded on the acellular matrix scaffolds by tissue culture in vitro. The structure of acellular matrix was analyzed by light microscopy and scanning electron microscopy. RESULTS: By treatment with 1% Triton X-100 for 84 h, the cells of thoracic aorta were fully come off and the three-dimensional structure of the matrix still remained. After modification by freeze-drying for 24 h and then thermal cross-linking under vacuum at 120 ℃ for 12 h, the tensile strength of the acellular matrix remarkable increased and reached the maximum breaking strength of 1.70 MPa. It was also showed that cultured endothelial cells grew on the surface of acellular matrix for 7 days and the typical structure of vessel- like intimal formation was observed under scanning electron microscopy. CONCLUSION: The acellular matrix and endothelial cells have favorable compatibility. The modified acellular matrix could be a good candidate scaffold for rebuilding the tissue engineering blood vessel.     

The impact of aortic bypass on hemodynamic changes caused by abdominal aorta occlusion

AIM: To investigate the impact of aortic bypass of different flow on hemodynamic changes caused by abdominal aorta occlusion. METHODS: A pig ischemic model was established by cross-clamping of the abdominal aorta above the celiac artery for 1 hour, and aortic bypass was used at the same time. The different flow of aortic bypass were placed between the proximal abdominal aorta and the left common iliac artery by using a roller pump. Two kinds of bypass flow rate were used with 50% and 70% of the volume of abdominal aorta flow cross-clamped site. The major hemodynamic changes were monitored during the different aortic cross-clamping and unlamping periods. RESULTS: All pigs without bypass (simple cross-clamping group) developed severe hemodynamic disturbance . Proximal arterial pressure rose markedly and distal arterial pressure dropped drastically during the period of cross-clamping, and there was a significant drop in the arterial pressure after unclamping. Heart rate increased obviously, and urine outputs decreased significantly compared with pre-clamping. The animals also developed a serious metabolic acidosis. In 70% flow group, the aforementioned values improved significantly when compared with that of 50% flow group. CONCLUSION: There was a severe hemodynamic alteration when the proximal abdominal aorta was cross-clamped for 1 hour, and the injury changes could be well prevented when the flow of aortic bypass was up to 70% of the volume of abdominal aorta flow.     

Study on VIP-immunoreactive nerve fibers in dental pulp- dentine complex of human premolar

AIM: To investigate the impact of aortic bypass of different flow on hemodynamic changes caused by abdominal aorta occlusion. METHODS: A pig ischemic model was established by cross-clamping of the abdominal aorta above the celiac artery for 1 hour, and aortic bypass was used at the same time. The different flow of aortic bypass were placed between the proximal abdominal aorta and the left common iliac artery by using a roller pump. Two kinds of bypass flow rate were used with 50% and 70% of the volume of abdominal aorta flow cross-clamped site. The major hemodynamic changes were monitored during the different aortic cross-clamping and unlamping periods. RESULTS: All pigs without bypass (simple cross-clamping group) developed severe hemodynamic disturbance . Proximal arterial pressure rose markedly and distal arterial pressure dropped drastically during the period of cross-clamping, and there was a significant drop in the arterial pressure after unclamping. Heart rate increased obviously, and urine outputs decreased significantly compared with pre-clamping. The animals also developed a serious metabolic acidosis. In 70% flow group, the aforementioned values improved significantly when compared with that of 50% flow group. CONCLUSION: There was a severe hemodynamic alteration when the proximal abdominal aorta was cross-clamped for 1 hour, and the injury changes could be well prevented when the flow of aortic bypass was up to 70% of the volume of abdominal aorta flow.     

Protective effect of bactericidal/permeability-increasing protein on sepsis induced by intra-abdominal infection in rats

AIM:To investigate the protective effect of bactericidal/permeability-increasing protein (BPI) on sepsis induced by intra-abdominal infection in rats and its mechanism.METHODS:Intra-abdominal infection induced sepsis was reproduced by cecal ligation and puncture (CLP). BPI or equal volume of physiological saline was intra-abdominally given immediately after CLP and 12 hours after CLP respectively (2.5 mg/kg of BPI each time). Plasma endotoxin levels were determined with limulus amebocyte chromogenic assay.RESULTS:(1)The survival time in BPI group was significantly higher than in physiological saline (PS) group. (2)The values of MAP, LVSP, IP, d p /d t max and-d p /d t max in BPI group, although decreasing,were markedly higher than those in PS group. (3) Plasma glutamate-pyruvate transaminase and urea nitrogen levels in BPI group, though increasing, were significantly lower than those in PS group.(4) There was no significant change of plasma endotoxin levels in BPI group, while plasma endotoxin levels were markedly increased in PS group. There was significantly different between two groups. CONCLUSIONS:BPI has an obvious protective effect on intra-abdominal infection induced sepsis, which might be related to its antagonism against endotoxin.