A non-human primate derived anti-P-selectin glycoprotein ligand-1 antibody curtails acute pancreatitis by alleviating the inflammatory responses

Acute pancreatitis (AP) is a devastating disease characterized by an inflammatory disorder of the pancreas. P-selectin glycoprotein ligand-1 (PSGL-1) plays a crucial role in the initial steps of the adhesive at process to inflammatory sites, blockade of PSGL-1 might confer potent anti-inflammatory effects. In this study, we generated two non-human primate derived monoclonal antibodies capable of efficiently targeting human PSGL-1, RH001-6 and RH001-22, which were screened from immunized rhesus macaques. We found that RH001-6, can effectively block the binding of P-selectin to PSGL-1, and abolish the adhesion of leukocytes to endothelial cells in vitro. In vivo, we verified that RH001-6 relieved inflammatory responses and pancreatic injury in both caerulein and l-arginine induced AP models. We also evaluated the safety profile after RH001-6 treatment in mice, and verified that RH001-6 did not cause any significant pathological damages in vivo. Taken together, we developed a novel non-human primate derived PSGL-1 blocking antibody with high-specificity, named RH001-6, which can interrupt the binding of PSGL-1 and P-selectin and attenuate inflammatory responses during AP. Therefore, RH001-6 is highly potential to be further developed into therapeutics against acute inflammatory diseases, such as AP.

Mechanistic study of β-tubulin and its interaction with the dopamine D5 receptor, cholecystokinin B receptor, and water-sodium metabolism / 中国比较医学杂志

Objective To examine the role of β-tubulin on the interaction between the cholecystokinin B receptor (CCKBR),dopamine D5 receptor(D5R), and water-sodium metabolism. Methods Normotensive and hypertensive renal proximal tubular cells(RPTC)were equally randomized into three separate groups: a gastrin group, fenoldopam group,and gastrin+nocodazole group. Immunofluorescence was used to determine localization of β-tubulin,CCKBR,and D5R. Western blotting was used to detect CCKBR, D5R, and Na-K-ATP expression. Results Gastrin stimulation in normotensive RPTC increased D5R expression(P < 0.05)and decreased Na-K-ATP expression(P < 0.05). These changes were blocked by a tubulin inhibitor(P < 0.05). However, interaction between CCKBR, D5R, and Na-K-ATP expression was not significantly affected in hypertensive RPTC. Immunofluorescence showed that CCKBR and D5R can induce one another,followed by transport to the plasma membrane, which can prevented by a tubulin inhibitor. Further, tubulin is disordered in hypertensive RPTC,which cannot support intracellular CCKBR and D5R transport. Conclusions tubulin plays a key role in the interaction between CCKBR, D5R, and water-sodium metabolism by improving protein transfer from the cytoplasm to cell membrane.

Protective effect and mechanism of Sestrin2 in the development of pulmonary alveolar type II epithelial cell injury induced by cigarette smoking / 中国比较医学杂志

Objective To explore the role of Sestrin2 in pulmonary alveolar type II epithelial cell injury induced by cigarette smoking and its mechanism of action. Methods The cell injury model was induced by cigarette smoke extract (CSE)in the human pulmonary alveolar type II epithelial A549 cells. The generation of ROS was detected by DCFDA fluorescence probe. The levels of inflammatory factors TNF-α and IL-8 were determined by ELISA, and the expression of Sestrin2 and the peroxiredoxin,Prx-SO2/3H,was detected by Western blot. In addition,all the events were also measured in the A549 cells which were transfected with Sestrin2 siRNA and treated with azithromycin. Results After the CSE treatment,the expression of Sestrin2 in the A549 cells was decreased, the expression of Prx-SO2/3H was increased, the ROS production,secretion of cytokines TNF-α and IL-8 were increased(P < 0.05). These changes were partly reducedby azithromycin, indicating that azithromycin significantly relieved CSE-induced oxidative stress and inflammatory injury.Silencing of Sestrin2 in the A549 cells result ed in an increase of Prx-SO2/3 H expression, ROS production and the secretionof the cytokines TNF-α and IL-8. However, oxidative stress and inflammatory injury were not alleviated with the addition ofazithromycin in the Sestrin2 siRNA silencing A549 cells. Conclusions Sestrin2 plays an protective role in the pulmonaryalveolar type II epithelial cell injury induced by cigarette smoking through negatively regulating the level of intracellularROS via catalyzing the reduction of the hyperoxidized peroxiredoxin Prx-SO2/3 H.

Study on the mechanism of PSGL-1 in salt-sensitive hypertension via inflammation in mice / 中国比较医学杂志

Objective To investigate the role and mechanism of PSGL-1 in development of salt-sensitive hypertension in mice. Methods PSGL-1 knockout(PSGL-1 -/-)and wild type(PSGL-1 +/ +)mice were fed a high salt (6% NaCl)or normal salt(0.4% NaCl)diet for three months. Blood pressure was measured under anesthesia via the carotid artery. The status of tissue inflammation and kidney injury was tested by flow cytometry, immunohistochemistry, and western blotting. Results Compared with mice fed a normal salt diet, PSGL-1 +/ +mice fed a high salt diet for three months showed high blood pressure, increased inflammatory cell infiltration in the aorta and skin, and increased inflammatory cytokine expression(interleukin-6, interleukin-1β, and tumor necrosis factor-α)in the kidney, as well as elevated expression of the kidney injury marker, connective tissue growth factor. In contrast, inflammation and kidney injury were not found in PSGL-1 -/-mice fed a high-salt diet. Conclusions In mice,PSGL-1 via inflammation plays a key role in development of hypertension and kidney injury caused by high salt intake.

The study on the mechanism of platelet in the development of salt-sensitive hypertension via inflammation / 中国比较医学杂志

Objective To investigate the role and mechanism of platelet in the development of salt-sensitive hypertension.Methods 25 Dahl salt-sensitive rats (Dahl SS) were divided into three groups: low-salt diet (0.12% NaCl, LS), high-salt diet (8%NaCl, HS) and high-salt diet + platelet inhibitor (8%NaCl+busulfan, HS+bus).Blood pressures were measured by tail-cuff method.After six weeks, animals were sacrificed.Platelet p-selectin expression, platelet cytosolic Ca2+ concentration, platelet-leukocyte aggregation (PLA) in peripheral blood, and immune cells infiltrated on aortic walls were assessed by flow cytometry, and serum IL-6 level was tested by ELISA in vivo.Platelets purified from SD rats were treated with normal salt (0.9%NaCl) and high salt (1.3%NaCl), then the cytosolic Ca2+ concentration and p-selectin expression of platelet were detected.Results We found that Dahl SS rats with high-salt diet, relative to low-salt diet, presented with high blood pressure and increased the ratio of platelet p-selectin expression, Ca2+ concentration.IL-6 level and PLA in peripheral blood, and the number of infiltrated immune cells on aortic walls were also significantly elevated in high-salt diet group.The whole events were ameliorated by the platelet inhibitor busulfan.Cytosolic Ca2+ concentration and p-selectin expression were also increased in purified platelets treated with high salt than those treated with low salt (P < 0.05).Conclusions Our findings suggest that high salt induced platelet activation with increased Ca2+ concentration may play an important role in the development of salt-sensitive hypertension via vascular inflammation.However, the detailed mechanisms of platelet activation and development of high blood pressure via inflammation induced by high salt intake remain to be determined.

Expression and relationship between hepaCAM protein and multidrug resistance protein in renal carcinoma / 国际肿瘤学杂志

Objective To investigate the expression and relationship between hepatocyte cell adhesion molecule (hepaCAM) protein and some multidrug resistance proteins in renal carcinoma tissue.Methods Expressions of hepaCAM,multidrug resistance associated protein (MRP),P-glycoprotein (P-gp),lung resistance protein (LRP),and topoisomerase Ⅱ (TOPO Ⅱ) protein were detected by immunohistochemistry in different areas of human renal cell carcinoma tissues and their relationships were analyzed.Results In the peripheral zone of renal tumor,hepaCAM,MRP,P-gp and LRP protein were showed positive expression.In the central region of the renal tumor,the expressions of hepaCAM,P-gp and LRP were negative or weakly positive,while the expressions of MRP and TOPO Ⅱ protein were positive.The expressions of MRP and TOPO Ⅱ protein in the central region of tumor were stronger than those in the peripheral zone of tunor (31.23 ±5.67 vs.23.89 ±4.56;45.66 ±2.34 vs.5.23 ±0.66),with statistically significant differences (t =-6.20,P =0.00;t =-100.16,P =0.00).While the expressions of other proteins (hepaCAM,P-gp and LRP) in the central region of tumor were weaker than those in the peripheral zone of tumor (3.21 ±1.12 vs.27.25±2.23;2.34±0.33 vs.51.23±3.45;4.22±1.78 vs.44.23 ± 1.45),with statistically significant differences (t =60.87,P =0.00;t =90.35,P =0.00;t =107.18,P =0.00).Correlation analysis showed that the expression of hepaCAM protein in the central region of renal carcinoma was related with the expression of MRP protein (r =0.94,P =0.01),but it was not related with the expressions of P-gp,LRP and TOPO Ⅱ protein (r=0.22,P=0.44;r=0.14,P=0.80;r=0.34,P=0.07).Conclusion The expression of hepaCAM protein in renal carcinoma may be related to tumor drug-resistance.

The regulation of dilated cardiomyopathy by dopamine D5 receptor through inhibiting oxidative stress / 中国比较医学杂志

Objective To determine whether dopamine D5 receptor (D5R) regulates the development of dilated cardiomyopathy (DCM) by inhibiting oxidative stress.Methods We developed heart-specific hD5 mutant (α-MHC-hD5F173L) and wild type (α-MHC-hD5WT) transgenic mice.The NOX2 expression and ROS production were tested in the transgenic mice at three month of age.The α-MHC-hD5F173L mice were treated with either NADPH oxidase inhibitor Apocynin (1mmol/kg/day) or phosphate-buffered saline (PBS) as control by intraperitoneal injection for 4 weeks.After then, the indexes of heart function were measured.The hD5WT and hD5F173L were transfected respectively in rat H9C2 cells, in which ROS production and NOX2 expression were detected at basal level.Results The ROS production and NOX2 expression were higher in the heart of α-MHC-hD5F173L than α-MHC-hD5WT mice.Apocynin treatment improved the heart function of α-MHC-hD5F173L mice.NOX2 expression and ROS production were higher in hD5F173L than hD5WT transfected H9C2 cells.Conclusions Dopomine D5 receptor may prevent DCM development by inhibiting oxidative stress.

The investigation of effect of trichostatin A to breast cancer MDA-MB-231 cell line / 重庆医科大学学报

Objective:To investigate the influence of histone deacetylase inhibitor(Trichostatin A)to multiplication,invasion, cell cycle of breast cancer MDA-MB-231 cell line.Methods:After 6-48hours treatment with the concentration(100～400nmol/L)of Trichostatin,the growth activity of MDA-MB-231 cell line was detected by MTT.The expression of ER?,MMP-9 mRNA was detected by RT-PCR with different concentration(100～400nmal/L TSA)during 24 hours to MDA-MB-231 cell line.The expression of ER?,MMP-9 protein was examined by immunohistochemistry with different concentration(100～400nmol/L TSA)during 24 hours treatment to MDA-MB-231 cell line.With cell invasion experiment to detect the change of invasive power by using different concentration(100～400nmol/L TSA)during 24 hours to MDA-MB-231 cell line.Results: Trichostatin can inhibit the growth of MDA-MB-231 cell line,which was time and dose dependent.Trichostatin can delay the cell cycle G_2/M stage,stop the cell stage in G_2 stage.Trichostatin can up-regulate the expression of ER?mRNA, down-regulate the expression of MMP-9 mRNA in cells.The invasion experiment indicate that Trichostatin can conspicuous inhibit the invasion of MDA-MB-231 cell line.Conclusions:Trichostatin can inhibit the accrementition,invasion and metastasis of MDA-MB-231 cell line evidently,and one of the mechanism may be the up-regulation of ER?and the down-regulation of MMP-9.