[ ABSTRACT] AIM:To investigate the protective effect of 1, 3-dicyclopentyl-1, 2, 3, 6-tetrahydropyrimidine-4, 5-dicarboxylic acid diethyl ester (ZL-5015) on lethal endotoxin-challenged mice and to explore the underlying mechanism. METHODS:Mouse model of lethal endotoxin challenge and endotoxemia were established by intraperitoneal administration of lipopolysaccharide (LPS) at a dose of 70 mg/kg to the C57BL/6J mice.Mouse peritoneal macrophages stimulated with LPS (10 mg/L) were used as an in vitro inflammatory model.The levels of interleukin-1β( IL-1β) , interleukin-10 ( IL-10) and tumor necrosis factor-α(TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA).Real-time PCR was used to evaluate the mRNA expression of the cytokines.RESULTS:Prophylactic treatment of the mice with ZL-5015 (100 and 200 mg/kg, ig) slightly increased the survival rate, extended the survival time, decreased the serum levels of IL-1βand TNF-α, and increased the serum level of IL-10 in the early stage of endotoxemia as compared with model group.The results of in vitro study demonstrated that treatment of the endotoxin-stimulated mouse peritoneal macrophages with ZL-5015 (10, 20 and 40μmol/L) inhibited the expression of IL-1βand TNF-αat both mRNA and protein levels but promoted the expression of IL-10 at both mRNA and protein levels.CONCLUSION: The tetrahydropyrimidine derivative ZL-5015 shows a moderate anti-endotoxin effect by increasing the survival rate and extending the survival time of the mice challenged by endotoxin, which may result from inhibition of the expression of pro-inflammatory cytokines such as IL-1βand TNF-α, and promotion of the expression of anti-inflammatory cytokine IL-10.

[ ABSTRACT] AIM:To investigate the effects of maternal limb ischemic preconditioning ( LIP) on the mitochon-drial structures and functions of the hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats. METHODS:Pregnant rats (n=40) were randomly divided into 4 groups: sham (S) group, LIP group, fetal distress ( FD) group and LIP+FD group.Intrauterine ischemia model was established through the experimental design.The ultra-structure of the mitochondria in CA1 area of the hippocampus was observed .The mitochondrial membrane potential and re-active oxygen species ( ROS) were measured .The content of ATP and MDA in the hippocampus tissue was detected.The activity of Mn-SOD was observed.RESULTS:Compared with sham group, the ultrastructure of mitochondria in CA1 area of the hippocampus was damaged in FD group and LIP+FD group.The mitochondrial membrane potential, the content of ATP and the activity of Mn-SOD were decreased.However, the content of ROS and MDA was increased.Compared with FD group, the ultrastructure of mitochondria in CA1 area of the hippocampus was intact in LIP+FD group.Furthermore, the reduced mitochondrial membrane potential and ATP content were inhibited.The activity of Mn-SOD was increased, but the content of ROS and MDA was decreased in LIP+FD group.CONCLUSION:Limb ischemia preconditioning inhibits the damage the mitochondria of fetal hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats.

[ ABSTRACT] AIM:To investigate the effects of cerebral ischemia and postconditioning on protein kinase R-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78) in the hippocampus tissue of tree shrew during endoplasmic reticulum stress and the mechanism of post-conditioning protecting the brain from damage.METH-ODS:The focal cerebral ischemic model was duplicated by photochemical reaction in tree shrew and the postconditioning was induced by alternatively occluding and opening the carotid artery of ischemic side for 3 cycles (5 min each cycle) at 3.5 h after ischemia.The damage and ultrastructural changes of the hippocampal neurons were observed by HE staining. The expression of PERK and GRP78 at mRNA and protein levels in the hippocampal tissue at different time points after cer-ebral ischemia and postconditioning was determined by RT-PCR, immunohistochemistry and Western blot.RESULTS:The injuries of hippocampal neurons were aggravated with prolonged cerebral ischemia, which was most severe at 24 h after ischemia while the postconditioning alleviated these damages correspondingly.The expression of PERK at mRNA and pro-tein levels was upregulated at 4 h, 24 h and 72 h after ischemia (P<0.05), while postconditioning downregulated the ex-pressions of PERK at ischemia and postconditioning 4 h (IP4 h) gruop and IP24 h group (P<0.05).The expression of GRP78 at mRNA and protein levels was not changed at 4 h, 24 h and 72 h after ischemia, while postconditioning upregu-
lated the expressions of GRP78 at IP24 h group (P<0.05).CONCLUSION:The focal thrombotic cerebral ischemia ac-tivates the endoplasmic reticulum stress in ischemic hippocampus of tree shrews, leading to the changes in mRNA and pro-tein expression of PERK in the PERK/eIF2αsignal transduction pathway.The postconditioning treatment alleviates endo-plasmic reticulum stress and neuronal damages by downregulating PERK and upregulating GRP78, thereby protecting the brain from injury.

[ ABSTRACT] AIM:To investigate the effects of microRNA145 ( miRNA145 ) on the viability, apoptosis, inva-sion and metastasis of hepatoma HepG2 cells.METHODS: HepG2 cells were randomly allocated into 3 groups: blank control group, empty mimic transfected group and miRNA145 mimic transfected group.Under the induction of Lipofectami-neTM 2000, the recombinant was transfected into HepG2 cells.After transfection, the expression level of miRNA145 was detected by real-time PCR.The protein level of N-cadherin and the mRNA expression levels of miRNA145 and N-cadherin were detected by Western blot and real-time PCR.The cell viability was detected by MTS assay.The cell cycle and apopto-sis were analyzed by flow cytometry.Invasion and metastasis were detected by Transwell assay.RESULTS:Compared with negative control, miRNA145 expression was up-regulated significantly, while the expression of N-cadherin was down-regu-lated significantly.Meanwhile, the cell viability, cell cycle, apoptosis, invasion and metastasis of hepatoma HepG2 cells were all significantly inhibited (P<0.05).CONCLUSION:miRNA145 dramatically inhibits viability, apoptosis, inva-sion and metastasis of hepatoma cells.

[ ABSTRACT] AIM:To study the effects of anti-aging Klotho protein on neonatal rat myocardial cells with hypo-xia/reoxygenation ( H/R) injury.METHODS:The cardiomyocytes of neonatal SD rats were cultured to establish hypoxia/reoxygenation model.The myocardial cells were divided into normal control group, H/R model group, different concentra-tions of Klotho protein (0.1μmol/L, 1μmol/L and 10μmol/L) pretreatment groups.The myocardial cells pulse frequen-cy was observed before and after H/R.The cell viability was measured by MTT assay.The leakages of LDH, CK and AST, the content of MDA and the activity of SOD were detected.The apoptotic rate of the myocardial cells was analyzed by flow cytometry.The mRNA expression of endoplasmic reticulum stress markers and apoptosis-related molecules GRP78, CRT, CHOP and caspase-12 was measured by real-time PCR.The protein levels of CHOP, caspase-12 and phosphorylated Akt in the myocardial cells were determined by Western blot.RESULTS: Compared with normal control group, the pulse fre-quency, cell viability rate and SOD activity of myocardial cells were significantly decreased, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were increased in H/R model group.The mRNA expressions of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and caspase-12 were increased, whereas p-Akt level was decreased obviously.Compared with H/R model group, the pulse frequency, cell viability rate and SOD activity were in-creased significantly, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were decreased in Klotho pretreated group.The mRNA expression of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and
caspase-12 were decreased, while p-Akt level increased significantly.CONCLUSION:Anti-aging Klotho protein improves the myocardial cell survival and inhibits the apoptosis by increasing the resistance of the cells to oxidative stress and exces-sive endoplasmic reticulum stress response, which is related with the activation of Akt phosphorylation in H/R-injured my-cardial cells.

[ ABSTRACT] AIM: To investigate the protective effect of mesenchymal stem cell ( MSC)-conditioned medium (MSCCM) on myocardial cell line H9c2 and its mechanism.METHODS:Verification of MSC was performed by flow cy-tometry analysis, followed by MTT assay to determine the optimal incubation time of MSCCM with myocardial cells.The cells were divided into 4 groups:normal ( N) group, model ( M) group, M+MSCCM group and MSCCM group.The cells in M+MSCCM group and MSCCM group were pre-incubated with MSCCM for 24 h.The cells in M group and M+MSCCM group were treated with 300 μmol/L H2 O2 for 4 h to imitate oxidative injury of myocardial cells.Mitochondrial membrane potential and apoptotic rate of injured myocardial cells were detected by flow cytometry.The ROS production was measured by fluorescence microscopy.The nuclear translocation of Nrf2 and expression of HO-1 was examined by Western blot.RE-SULTS:No difference of mitochondrial membrane potential, apoptotic rate or ROS production between MSCCM group and N group was observed (P>0.05).The mitochondrial membrane potential depolarization, apoptotic rate and ROS produc-tion in M+MSCCM group were significantly lower than those in M group ( P<0.01 ) .The nuclear translocation of Nrf2 and expression of HO-1 in the myocardial cells were increased with MSCCM incubation time prolonged.CONCLUSION:MSCCM protects the myocardial cells against oxidative injury induced by H2 O2 .The anti-oxidative mechanism would be as-sociated with the activation of Nrf2/ARE pathway.

[ ABSTRACT] AIM:To establish a perforated whole-cell patch-clamp technique withβ-escin to record L-type cal-cium current (ICa,L) in osteoblasts.METHODS:ROS 17/2.8 is a rat osteoblast-like osteosarcoma cell line.β-escin was applied to the pipette solution to permeabilize the cell membrane and the perforated patch recording mode was obtained. RESULTS:β-escin at concentration of 50μmol/L easily permeabilized the cell membrane and obtained a perforated patch recording mode in 2~7 min.This technique prevented ICa,L rundown and preserved cytoplasmic signaling pathways.CON-CLUSION:β-escin may be used as an alternative ionophore for perforated patch-clamp studies in osteoblasts and results in minimal rundown that could facilitate recordings of ICa,L in osteoblasts.

Protein disulfide isomerase A3 (PDIA3) is a member of protein disulphide isomerase family and widely exists in endoplasmic reticulum, cell surface, nucleus and mitochondria.PDIA3 promotes the glycoprotein folding and quality control in the endoplasmic reticulum and is also a key molecular of major histocompatibility complex class I mol-ecules assembly.In addition, PDIA3 is involved in the cell signal transduction and plays an important role in a variety of disease development.Therefore, this paper talks about the function of PDIA3, the relationship between disease and PDIA3 as well as its clinical outlook.

[ ABSTRACT] AIM:To observe the effect of ursodeoxycholic acid ( UDCA) on the treatment of infantile hepatitis syndrome ( HIS) and to investigate its mechanism.METHODS:The children with infantile hepatitis syndrome were divid-ed into conventional treatment group and the UDCA treatment group.Twenty healthy children were selected as normal con-trol.The children in conventional therapy group were given antiviral and hepatoprotective treatments.The children in UD-CA treatment group were given ursodeoxycholic acid (10 mg? kg-1? d-1 ) in addition to the conventional treatment group for 2 to 3 weeks.The levels of total bilirubin (TBIL), direct bilirubin (DBIL), alanine aminotransferase (ALT), glu-tamyltransferase ( GGT) , total bile acids ( TBA) and TNF-α, IL-6 were detected before admission and 2 weeks later.RE-SULTS:The levels of TNF-αand IL-6 were significantly higher in the children with IHS than those in the normal control (P<0.01).The levels of TBIL, DBIL, ALT, GGT, TBA, TNF-αand IL-6 in conventional treatment group were reduced after therapy (P<0.01).All the above index in UDCA treatment group were decreased compared with conventional treat-ment group (P<0.01).CONCLUSION:On the basis of conventional therapy, ursodeoxycholic acid effectively alleviates the systemic inflammatory response in the children with IHS, reduces the liver damages.

[ ABSTRACT] AIM:In order to observe the myocardial differentiation capacity of the dedifferentiated fat ( DFAT) cells treated with vitamin C in vitro.METHODS: DFAT cells were dedifferentiated from the mature rat adipocytes with ceiling adherent culture.The DFAT cells of passage 3 were used in the study.Vitamin C and/or neonatal rat heart tissue lysate were added into the culture medium to induce myocardial differentiation for 3 weeks.The cell morphology was ob-served under microscope.The myocardial-specific markers, such as cTnT, GATA-4 and NKx2.5, were examined by the methods of immunofluorescence, PCR and Western blot.RESULTS:Mature rat adipocytes dedifferentiated into fibroblast-like DFAT cells after ceiling adherent culture.The DFAT cells spontaneously differentiated into cardiomyocyte-like cells under normal culture condition with a low incidence.After treated with neonatal rat heart cell lysate, the DFAT cells be-came cardiomyocyte-like cells that had bigger size, longer shape and myotubule-structure.The expression of cTnT, GATA-4 and NKx2.5 was remarkably increased at both mRNA and protein levels as compared with the normal cultured DFAT cells.The expression of cTnT, GATA-4 and NKx2.5 was further increased in DFAT cells after treating with vitamin C.No spontaneous beating cell was observed.CONCLUSION:Vitamin C enhances the differentiation of DFAT cells into cardio-myocyte-like cells.