ObjectiveTo establish the characteristic sugar spectrum of polysaccharides， oligosaccharides and monosaccharides of wild-simulated and transplanted Astragali Radix， and find out the difference of the sugar spectrum between the two， so as to provide a basis for quality evaluation of Astragali Radix. MethodThe relative molecular weight distribution of polysaccharides from 18 batches of wild-simulated Astragali Radix and 12 batches of transplanted Astragali Radix were characterized by high performance liquid chromatography-evaporative light scattering detection（HPLC-ELSD） to establish the characteristic chromatograms of two kinds of polysaccharides. The difference in the peak area ratio of APS-Ⅱ， a polysaccharide component with a relative molecular weight of 10 kDa， in two kinds of Astragali Radix was analyzed， and the critical value of peak area ratio of APS-Ⅱ was determined by receiver operating characteristic（ROC） curve. At the same time， APS-Ⅱ was partially acid-hydrolyzed by trifluoroacetic acid（TFA） to establish characteristic spectra of two kinds of oligosaccharides from Astragali Radix based on HPLC-ELSD， and the characteristics of differential oligosaccharides were found by principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）. Two kinds of APS-Ⅱ were completely acid-hydrolyzed by TFA and derivatized to establish characteristic spectra of two kinds of monosaccharides from Astragali Radix based on HPLC， PCA and OPLS-DA were performed on the peak area ratio of two kinds of monosaccharides to explore the differences in the composition of two kinds of APS-Ⅱ monosaccharides. ResultThe characteristic sugar spectrum of polysaccharides from Astragali Radix showed that the peak area ratio of APS-Ⅱ was the main difference， and the peak area of APS-Ⅱ of wild-simulated and transplanted Astragali Radix were 89.17%-97.17% and 80.14%-91.96%， respectively. The ROC curve determined the critical value of 92.28% for the difference of APS-Ⅱ peak area ratio of the two kinds of Astragali Radix. The multivariate analysis of APS-Ⅱ oligosaccharides revealed that the peak area ratio of oligosaccharides with polymerization degree≥10 was the main difference， which ranged from 11.835%-19.092% for wild-simulated products and 2.778%-7.017% for transplanted products. The results of monosaccharide characteristic sugar spectrum analysis showed that both Astragali Radix species consisted of six monosaccharides， and glucose and arabinose were the differential monosaccharide fractions. The peak area ratios of glucose and arabinose in wild-simulated products were 85%-93.9% and 2.7%-5.8%， respectively， while those of transplanted products were 74.3%-87.3% and 5.3%-10.7%， suggesting that the structures of the two polysaccharide fractions APS-Ⅱ of Astragali Radix may be different. ConclusionThe difference of sugar spectrum between two kinds of Astragali Radix may be related to the content and structure of APS-Ⅱ， and this study may provide a reference for the study of carbohydrates in Astragali Radix and the quality evaluation of medicinal materials.

Objective:To evaluate the role of TANK-binding kinase-1 (TBK1) in renal fibrosis in mice with acute kidney injury (AKI) and relationship with endoplasmic reticulum stress.Methods:Twenty-four male wild-type C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group CON), group AKI, control plus TBK1 inhibitor group (group CON-GSK) and AKI plus TBK1 inhibitor group (group AKI-GSK). In group AKI and group AKI-GSK, folic acid 250 mg/kg was intraperitoneally injected to prepare AKI model.From the first day after folic acid injection, 1% dimethyl sulfoxide 20 ml/kg was intraperitoneally injected every other day in group AKI, and GSK8612 1.5 mg/kg was intraperitoneally injected every other day in group AKI-GSK, 7 times in total.In group CON and group CON-GSK, 1% dimethyl sulfoxide 20 ml/kg and GSK8612 1.5 mg/kg were intraperitoneally injected, respectively, every other day for 7 times in total.On the 14th day after injection of folic acid, the eyeball blood samples were taken to determine the concentrations of serum blood urea nitrogen (BUN) and creatinine (Cr), and the kidney tissues were also extracted, and the pathological results of renal tissue were observed by Sirius red staining, Masson staining and HE staining.The area of renal fibrosis was measured and the tubulointerstitial injury score was calculated.The expression of fibronectin, type I collagen and α-smooth muscle actin was detected by immunofluorescence.The expression of phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), activated transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), phosphorylated inositol-requiring kinase 1α (p-IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), apoptosis-regulating signal kinase 1 (ASK1), caspase-12 and phosphorylated c-Jun N-terminal protein kinase (p-JNK) was detected by Western blot. Results:Compared with group CON, the serum BUN and Cr concentrations, area of renal fibrosis and renal tubulointerstitial injury score were significantly increased, and the expression of fibronectin, type I collagen, α-smooth muscle actin, p-PERK, p-eIF2α, ATF4, CHOP, p-IRE1α, TRAF2, ASK1, caspase-12, and p-JNK was up-regulated in group AKI and group AKI-GSK ( P<0.05), and no significant change was found in the indexes mentioned above in group CON-GSK ( P>0.05). Compared with group AKI, the serum BUN and Cr concentrations, area of renal fibrosis, and tubulointerstitial injury score were significantly decreased, and the expression of fibronectin, type I collagen, α-smooth muscle actin, p-PERK, p-eIF2α, ATF4, CHOP, p-IRE1α, TRAF2, ASK1, caspase-12, and p-JNK was down-regulated in group AKI-GSK ( P<0.05). Conclusions:TBK1 is involved in the process of renal fibrosis in mice with AKI, and the mechanism may be related to the promotion of endoplasmic reticulum stress.

Objective:To evaluate the immunological efficacy of a novel DNA vaccine against West Nile virus (WNV) in a mouse model.Methods:A DNA vaccine VRC-prME expressing the precursor membrane (prM) and envelope protein (E) of WNV Xinjiang strain (XJ11129-3) was constructed and its ability to express virus-like particles was verified in vitro. C57BL/6 mice were immunized twice with VRC-prME via intramuscular injection combined with electroporation with an interval of four weeks. Enzyme-linked immunoassay (ELISA) was used to detect serum antibodies after immunization. WNV (NY99 strain) single-round infectious particles were used to detect neutralizing antibodies. Cellular immune responses were analyzed by enzyme-linked immunoblot assay (ELISPOT) and intracellular cytokine staining (ICS). Results:VRC-prME induced a strong Th1-biased antibody response in mice that could cross-neutralize the WNV (NY99 strain) single-round infectious particles two weeks after the boost immunization. Moreover, the vaccine also elicited antigen-specific multifunctional CD8 + T cell responses (IFN-γ, IL-2, TNF-α). Conclusions:The novel DNA vaccine prepared in this study, expressing the prME protein of WNV XJ11129-3 strain, could induce stronger humoral and cellular immune responses in mice, which was worthy of further research and development for the prevention of WNV infection in China.

[Objective] To explore whether necroptosis is involved in the mechanism of lung injury induced by intestinal ischemia-reperfusion.[Method] Thirty-two healthy male Sprague-Dawley rats were randomly assigned into 4 groups (n--8):sham operation group (sham group),isehemia/ reperfusion group (I/R group),necroptosis inhibitor necrostatin-1 group (Nec-1 group) and solvent dimethyl sulfoxide (DMSO) group (DMSO group).Model of intestinal I/R injury was produced by clamping the superior mesenteric artery for 1.5 h followed by 6 h reperfusion in rats.Necrostatin-1 1.0 mg/kg was administered 30 min before occlusion in Nec1 group,while the equal volume of DMSO was given instead in DMSO group.The rats were sacrificed at 6 h of reperfusion and the lung tissues were removed for measurement of wet-dry ratio and microscopic examination and scored.The expression of receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3) in lung tissues was detected using Western-blot and immunohistochemistry.[Result] Compared with sham group,lung morphology score and wet/dry ratio in I/R,DMSO group raised (P ＜ 0.05).Lung morphology score and wet/dry ratio statistically declined in Nec-1 group compared with I/R and DMSO group (P ＜ 0.05),while there was no statistical difference of wet/dry ratio between sham group and Nec-1 group (P ＞ 0.05).As the result of westernblot and immunohistochemistry showed,the expression of RIP1 and RIP3 was up-regulated in I/R group and DMSO group (P ＜0.05),which was inhibited by Nec-1 in Nec-1 group (P ＜ 0.05).[Conclusion] Necroptosis is involved in the mechanism of lung injury induced by intestinal ischemia-reperfusion,and Nec-1,the special inhibitor of RIP1,can reduce the injury.

Objective To evaluate the role of necroptosis in liver injury induced by intestinal ischemia-reperfusion (I/R) in rats.Methods Thirty-two healthy adult male Sprague-Dawley rats,weighing 250-300 g,were divided into 4 groups (n=8 each) using a random number table:sham operation group (S group),I/R group,specific necroptosis inhibitor necrostatin-1 group (N group) and dimethyl sulfoxide (DMSO) group (D group).Intestinal I/R was produced by occlusion of the superior mesenteric artery for 1.5 h followed by 6 h of reperfusion.The superior mesenteric artery was only isolated but not ligated in group S.At 30 min before ischemia,necrostatin-1 1 mg/kg (diluted to 200 μl in DMSO) was intraperitoneally injected in group N,while the equal volume of DMSO was given instead in group D.The animals were sacrificed at the end of reperfusion,livers were removed for examination of the pathological changes with a light microscope,and the severity of liver injury was evaluated using the Eckhoff's scale score.Blood samples were collected from the cardiac apex for determination of serum alanine transaminase (ALT) concentrations by enzyme-linked immunosorbent assay.The expression of receptor-interacting protein kinase 1 (RIP1),RIP3 and high-mobility group box 1 protein (HMGB1) in cytoplasm of hepatocytes was detected by Western blot.The location of RIP1 and RIP3 in liver tissues was determined by immunohistochemistry,and the translocation of HMGB1 from nucleus to cytoplasm was tested by immunofluorescence.Results Compared with group S,the Eckhoff's scale score of liver tissues and serum ALT concentration were significantly increased,the expression of RIP1,RIP3 and HMGB1 in liver tissues was up-regulated (P＜0.05),and the hepatocytes in which RIP1 and RIP3 were highly expressed in the portal area were increased in group I/R.Compared with group I/R,the Eckhoff's scale score of liver tissues and serum ALT concentration were significantly decreased,the expression of RIP1,RIP3 and HMGB1 in liver tissues was down-regulated (P＜0.05),and the hepatocytes in which RIP1 and RIP3 were highly expressed in the portal area were decreased in group N,and no significant changes were found in the variables mentioned above in group D (P＞0.05).HMGB1 was expressed in the nucleus of hepatocytes in the portal area in group S;a large number of HMGB1 in hepatocytes in the portal area was translocated to cytoplasm in I/R and D groups;a small number of HMGB1 in hepatocytes in the portal area was translocated to cytoplasm in group N.Conclusion Necroptosis is involved in intestinal I/R-induced liver injury in rats.

Objective To evaluate the role of necroptosis in intestinal ischemia-reperfusion (I/R) injury in rats.Methods Thirty-two healthy male Sprague-Dawley rats,weighing 200-220 g,were randomly assigned into 4 groups (n =.8 each) using a random number table:sham operation group (Sham group),I/R group,necroptosis inhibitor necrostatin-1 group (Nec-1 group) and solvent dimethyl sulfoxide (DMSO) group (group DMSO).Intestinal I/R injury was produced by clamping the superior mesenteric artery for 1 h followed by 24 h reperfusion in rats anesthetized with chloral hydrate.Necrostatin-1 1.0 mg/kg was administered intraperitoneally at 30 min before occlusion in Nec-1 group,while the equal volume of DMSO was given instead in group DMSO.The rats were sacrificed at 24 h of reperfusion and the intestinal tissues were removed for microscopic examination.Intestinal damage was assessed and scored according to Chiu.Blood samples were taken for determination of serum diamine oxidase (DAO) activity.The expression of activitied caspase-3 and receptor-interacting protein 1 (RIP1) in intestinal tissues was detected using Western blot.Results Compared with Sham group,Chiu's score,serum DAO activity,and the expression of activitied caspase-3 and RIP1 was up-regulated in I/R,DMSO and Nec-1 groups.Compared with I/R and DMSO groups,Chiu's score and DAO activity were significantly decreased,the expression of RIP1 was down-regulated,and no significant change was found in the expression of activitied caspase3 in group Nec-1.Conclusion Necroptosis is involved in intestinal I/R injury in rats.

Aim To investigate the the protective effects of a novel recombinant Trichinella spiralis 38 ku protein ( rTsP38 ) on intestinal I/R injury and the po-tential mechanisms. Methods Male BALB/c mice were randomly divided into sham group ( group S) , in-jury group ( group I) , rTsP38 vaccinated group ( group T) and adjuvants vaccinated group ( group A ) , and received subcutaneously phosphate buffer solution (PBS), PBS, rTsP38, or adjuvants, respectively, at 2-week intervals 6 weeks before the surgical proce-dure. Results Intestinal I/R caused severe intestinal injury evidenced by significant increases in modified Chiu 's score and neutrophils infiltration, accompanied by decreases in daily food intake and body weight. The mRNA level of arginase-1 ( Arg-1 ) was decreased and the mRNA level of inducible nitric oxide synthase 2 ( NOS2) was increased in group I. RTsP38 significant-ly ameliorated intestinal injury and improved intestinal function following intestinal I/R accompanied by de-crease in neutrophils infiltration and increase in cell proliferation in the intestine, compared to mice without rTsP38 pretreatment. Fold changes of Arg-1 mRNA level were significantly increased in group T. Conclu-sions These findings indicate that rTsP38 exerts pro-tection on intestinal I/R injury in mice via promoting M2 macrophages polarization.

Objective To investigate the effects of remote limb ischemic preconditioning (RLIP) on the lung injury in patients undergoing abdominal aortic aneurysm repair.Methods Sixty-two ASA Ⅱ or Ⅲ patients of both sexes,aged 54-72 yr,with body mass index 21-36 kg/m2,undergoing elective abdominal aortic aneurysm repair,were randomly divided to 2 groups ( n =31 each):control group (group C) and RLIP group.RLIP consisted of two 5-min cycles of left upper limb ischemia induced by a blood pressure cuff placed on the left upper arm and inflated to 200 mm Hg,with an intervening 5 min of reperfusion,during which time the cuff was deflated.RLIP was performed after anesthesia induction and before the start of surgery.Arterial and venous blood samples were taken at 10 min after intubation (T0),and 30 min and 4,8,12 and 24 h after aortic unclamping (T1-5) for blood gas analysis and determination of the concentrations of serum interleukin (IL)-6,tumor necrosis factor (TNF)-α,and plasma malondialdehyde (MDA) and superoxide dismutase (SOD) activity.The alveolar-arterial oxygen pressure difference (PA-aO2 ) and respiratory index (RI) were calculated.The peak airway pressure (Ppeak),plat airway pressure (Pplat) and positive end expiratory pressure (PEEP) were recorded at the same time points mentioned above to calculate dynamic lung compliance (Cd) and static lung compliance (Cs).The incidence of hypoxemia,extubation time and duration of stay in intensive care unit (IGU) were also recorded.Results Compared with group C,PA-aO2,RI and the concentration of IL-6 were significantly decreased at T3-5,Cs,Cd and SOD activity were significantly increased at T2-5,and the concentrations of TNF-α and MDA were significantly decreased at T2-5 in group RLIP ( P ＜ 0.05).Compared with group C,the incidence of hypoxemia was significantly decreased,and extubation time and duration of stay in ICU were significantly shortened in group RLIP ( P ＜ 0.05).Conclusion RLIP can reduce the lung injury through inhibition of the inflammatory response and lipid peroxidation in patients undergoing abdominal aortic aneurysm repair.

ObjectiveTo investigate the role of 15-F2t-isoprostane in intestinal injury induced by intestinal ischemia/reperfusion (I/R) in rats.MethodsThirty-two pathogen free adult male SD rats weighing 230-255 g were randomly divided into 4 groups ( n =8 each):group sham operation (group S) ; group intestinal I/R; group SQ-29548 (TXA2 receptor antagonist) (group SQ) and group DMSO (the solvent).Intestinal I/R was induced by 60 min occlusion of superior mesenteric artery (SMA) followed by 120 main reperfusion in groups I/R,SQ and DMSO SQ-29548 2 μmol/kg and DMSO were injected subcutaneusly at abdominal wall at 30 min before SMS in groups SQ and DMSO respectively.Arterial blood samples were taken at 120 min of reperfusion for determination of serum diamine oxidase (DAO) activity and 15-F2t-isoprostane,endothelin-1 (ET-1) and thromboxane B2 (TXB2) concentrations.Intestinal tissues were removed for microscopic examination and determination of myeloperoxidase (MPO) and SOD activities,MDA and lactate contents.Intestinal damage was assessed and scored according to Chiu (0 =normal,5 =disruption of tunica propria,bleeding and ulceration).ResultsIntestinal I/R significantly increased Chiu's score,MDA and lactate contents and MPO activity and decreased SOD activity in intestine in group I/R as compared with group S.SQ-29548 pretreatment significantly decreased Chiu's score,lactate content and MPO activity in intestine and increased intestinal SOD activity and decreased serum DAO activity and ET-1 concentration in group SQ as compared with group I/R.Conclusion15-F2t-isoprostane is involved in the development of intestinal injury induced by intestinal I/R by activating TXA2 receptor,increasing ET-1 production and promoting neutrophil infiltration.

Objective To investigate the effects of intestinal ischemia-reperfusion (I/R) on the brain in rats. Methods Sixty-four healthy male SD rats weighing 250-300 g were randomly allocated to one of 2 groups (n = 32 each): sham operation group (S) and intestinal I/R group (I/R). Intestinal I/R was produced by occlusion of superior mesenteric artery (SMA) for 90 min followed by reperfusion. Eight animals were sacrificed at each of the following time points: 2, 6, 12 and 24 h of reperfusion (T1-4) in each group. After a median sternotomyblood samples were taken from left ventricle for measurement of plasma TNF-α and IL-6 (by ELISA). Intestine and brain tissue was harvested for microscopic examination and detection of apoptosis ( by TUNEL). The cognitive function was tested using Morris water maze at 24 h. Results No abnormality was found in intestine and brain tissue in group S. Intestinal damage and neurodegeneration were detected in group I/R. Intestinal I/R significantly increased cerebral apoptosis in group I/R compared with group S. Plasma TNF-a and IL-6 concentrations were significantly higher at T1-4 in group I/R than in group S. The escape latency and swimming distance were significantly increased, while the number of crossing the platform was decreased in group I/R compared with group S. There was no significant difference in the swimming speed between the 2 groups. Conclusion Intestinal I/R can induce brain injury and lead to cognitive dysfunction. I/R-induced release of inflammatory mediators and neuronal apoptosis are involved in the underlying mechanism.