Protective Effects of NaHS/miR-133a-3p on Lipopolysaccharide-Induced Cardiomyocytes Injury.

Objective: The aim of this study was to investigate the effects of sodium hydrosulfide (NaHS) on Lipopolysaccharide (LPS)-induced cardiomyocyte injury in H9c2 cells. Methods: H9c2 cardiomyocytes cultivated with medium containing 10 µg/mL LPS were used to recapitulate the phenotypes of those in sepsis. Two sequential experiments were performed. The first contained a control group, a LPS group, and a LPS + NaHS group, with the aim to assure the protective effects of NaHS on LPS-treated cardiomyocytes. The second experiment added a fourth group, the LPS + NaHS + miR-133a-3p inhibition group, with the aim to preliminarily explore whether miR-133-3p exerts a protective function downstream of NaHS. The adenosine triphosphate (ATP) kit was used to detect ATP content; real-time quantitative polynucleotide chain reaction (qPCR) was used to measure the levels of mammalian targets of rapamycin (mTOR), AMP-dependent protein kinase (AMPK), and miR-133a-3p, and Western blot (WB) was used to detect protein levels of mTOR, AMPK, myosin-like Bcl2 interacting protein (Beclin-1), microtubule-associated protein 1 light chain 3 (LC3I/II), and P62 (sequestosome-1, sqstm-1/P62). Results: Compared with the control group, the expressions of miR-133a-3p (P < 0.001), P62 (P < 0.001), and the content of ATP (P < 0.001) decreased, while the expressions of Beclin-1 (P = 0.023) and LC3I/II (P = 0.048) increased in the LPS group. Compared with the LPS group, the expressions of miR-133a-3p (P < 0.001), P62 (P < 0.001), and the content of ATP (P < 0.001) in the NaHS + LPS group increased, while the expressions of Beclin-1 (P = 0.023) and LC3I/II (P = 0.022) decreased. Compared with the NaHS + LPS group, the expression levels of miR-133a-3p (P < 0.001), P62 (P = 0.001), and the content of ATP (P < 0.001) in the LPS + NaHS + miR-133a-3p inhibition group were downregulated, and the expression levels of Beclin-1 (P = 0.012) and LC3I/II (P = 0.010) were upregulated. The difference was statistically significant. There was no significant difference in the expression of AMPK and mTOR between groups. Conclusion: Our research demonstrated that NaHS relieved LPS-induced myocardial injury in H9c2 by promoting the expression of miR-133a-3p, inhibiting autophagy in cardiomyocytes, and restoring cellular ATP levels.

MCC950 Ameliorates Acute Exogenous Lipoid Pneumonia Induced by Sewing Machine Oil in Rats via the NF-κB/NLRP3 Inflammasome Pathway.

BACKGROUND/AIM: Acute exogenous lipoid pneumonia (AELP) is a rare disorder caused by intake of lipid formulations and is often underdiagnosed. Meanwhile, the mechanism of AELP is still underlying. MCC950, was previously found to significantly suppress the release of inflammatory cytokines IL-18 and IL-1ß. However, the effect of MCC950 on AELP induced by sewing machine oil has not been reported. MATERIALS AND METHODS: The NLRP3, NF-[Formula: see text]B p65, caspase-1 and IL-1ß expression in lung tissues were compared between a rat model of AELP and control rats using western blotting and real-time quantitative assay. Moreover, haematoxylin and eosin (H&E) staining was performed to elucidate the mechanisms by which MCC950 ameliorates sewing machine oil-induced AELP in vivo. RESULTS: MCC950 reduced the expression of NF-[Formula: see text]B p65 in the lung samples of the treatment group and further down-regulated the NLRP3 and caspase-1 levels while inhibited the production of IL-1ß. Besides, decreases in inflammatory cell infiltration in the lung were shown using H&E staining. CONCLUSION: MCC950 ameliorates sewing machine oil-induced acute exogenous lipoid pneumonia in rats through inhibition of the NF-[Formula: see text]B/NLRP3 inflammasome pathway.

Dusp1 regulates thermal tolerance limits in zebrafish by maintaining mitochondrial integrity.

Temperature tolerance restricts the distribution of a species. However, the molecular and cellular mechanisms that set the thermal tolerance limits of an organism are poorly understood. Here, we report on the function of dual-specificity phosphatase 1 (DUSP1) in thermal tolerance regulation. Notably, we found that dusp1 -/- zebrafish grew normally but survived within a narrowed temperature range. The higher susceptibility of these mutant fish to both cold and heat challenges was attributed to accelerated cell death caused by aggravated mitochondrial dysfunction and over-production of reactive oxygen species in the gills. The DUSP1-MAPK-DRP1 axis was identified as a key pathway regulating these processes in both fish and human cells. These observations suggest that DUSP1 may play a role in maintaining mitochondrial integrity and redox homeostasis. We therefore propose that maintenance of cellular redox homeostasis may be a key mechanism for coping with cellular thermal stress and that the interplay between signaling pathways regulating redox homeostasis in the most thermosensitive tissue (i.e., gills) may play an important role in setting the thermal tolerance limit of zebrafish.

Multilocus sequence analyses reveal extensive diversity and multiple origins of fluconazole resistance in Candida tropicalis from tropical China.

Candida tropicalis is among the most prevalent human pathogenic yeast species, second only to C. albicans in certain geographic regions such as East Asia and Brazil. However, compared to C. albicans, relatively little is known about the patterns of genetic variation in C. tropicalis. This study analyzed the genetic diversity and relationships among isolates of C. tropicalis from the southern Chinese island of Hainan. A total of 116 isolates were obtained from seven geographic regions located across the Island. For each isolate, a total of 2677 bp from six gene loci were sequenced and 79 (2.96%) polymorphic nucleotide sites were found in our sample. Comparisons with strains reported from other parts of the world identified significant novel diversities in Hainan, including an average of six novel sequences (with a range 1 to 14) per locus and 80 novel diploid sequence types. Most of the genetic variation was found within individual strains and there was abundant evidence for gene flow among the seven geographic locations within Hainan. Interestingly, our analyses identified no significant correlation between the diploid sequence types at the six loci and fluconazole susceptibility, consistent with multiple origins of fluconazole resistance in the Hainan population of C. tropicalis.

[The research applications of db/db mouse].

The db/db mice are perfect animal models of type 2 diabetes which have been widely used. The phenotypes of severe obesity, hyperphagia, polydipsia, and polyuria are due to a spontaneous mutation of the leptin receptor (Lepr). The course of the disease is markedly influenced by genetic background, which is more serious in the C57BLKS/J background. And there are many other spontaneous mutations in different sites of Lepr, which produce a series of animal models of obesity, including db(3J)/ db(3J) mice, db(5j)/db(5J) mice, db(pas)/db(pas) mice, Zucker fa/fa rats, and Koletsky fa(k)/fa(k) rats, etc. These rodents appear similar hyperphagia and severe obesity, but different levels of blood glucose, kidney damage and reproductive ability, providing profuse material to investigate the complex function of Lepr. Here we review the history of the discovery of the leptin signaling pathway, the abnormal phenotypes of db/db mice in metabolic, reproductive, immune, etc. Discuss their research applications, reproductive strategy, genotyping guideline, the phenotypic diversity of those animal models with Lepr spontaneous mutation and their mutation patterns, respectively.

[Construction of the skeletal muscle-specific TbetaR II knockout mice].

OBJECTIVE: To generate the skeletal muscle-specific transforming growth factor beta receptor II (TbetaR II) gene knockout mice for the research on the function of the TbetaR II gene in skeletal muscles. METHODS: TbetaR II (flox/flox) mice were generated using embryonic stem cell technology. The MCK-Cre mice were engineered containing Cre recombinase under the control of creatine kinase (MCK) muscle-specific promoter. TbetaR II (flox/flox) mice were crossed with MCK-Cre mice generating TbetaR II (flox/flox)/MCK-Cre double Tg mice. And then, TbetaR II (flox/wt) /MCK-Cre(+) double Tg mice were crossed with TbetaR II (flox/flox) mice to generate TbetaR II (flox/flox)/MCK- Cre(+) mice genetically ablating TbetaR II in cre-expressing skeletal muscle cells. RESULTS: As predicted, mice lacking TbetaR II by gene targeting in skeletal muscles were generated first in the world using Cre/loxP system. TbetaR II null mutant mice were viable, fertile and showed apparently normal development.

Chemoresistance to temozolomide in human glioma cell line U251 is associated with increased activity of O6-methylguanine-DNA methyltransferase and can be overcome by metronomic temozolomide regimen.

Temozolomide (TMZ) is a novel cytotoxic alkylating agent for chemotherapy of malignant gliomas. However, intrinsic or acquired resistance to TMZ often defines poor efficacy of chemotherapy in malignant gliomas. A growing number of studies indicate that expression of O(6)-methylguanine-DNA methyltransferase (MGMT) is one of the principal mechanisms responsible for this chemoresistance. In the present study, we evaluated the relationship between expression of MGMT and resistance to TMZ. We generated a TMZ-resistant cell line, U251/TR, by stepwise (8 months) exposure of parental U251 cells to TMZ. The resistance to TMZ was quantified using SRB assay. MGMT expression was evaluated at mRNA (RT-PCR) and protein (Western blot) levels. U251/TR cells showed increased (~ sevenfold) resistance to TMZ. The MGMT expression (both mRNA and protein) was significantly (P < 0.01) increased in U251/TR cells compared with parental U251 cells. Further, MGMT expression fluctuated during exposure of U251/TR cells to TMZ. The resistance of U251/TR cells to TMZ could be overcome by application of elevated doses of TMZ when MGMT expression was at the lowest level. In conclusion, our results demonstrate that the primary mechanism responsible for resistance of U251/TR cells to TMZ is associated with increased expression of MGMT. Resistance of malignant gliomas to TMZ can be overcome by synchronizing metronomic TMZ regimen with MGMT expression.

[Correlation between the distribution of CD133-positive cells and the proliferation of microvessels in glioblastoma multiforme].

OBJECTIVE: To investigate the expression of CD133 and CD34 in different parts of glioblastoma and its margin and explore the invasive path of glioma stem cells within the tumor and surrounding tissue. METHODS: The surgical specimens were collected from the core of mass, junctional zones between tumor and peritumoral edematous areas and edematous areas in 52 patients with glioblastoma. Immunohistochemical cell staining and Western blot were employed to evaluate the expression of CD133 in different specimens while immunohistochemistry was used to detect the CD34-microvessel postforming. A correlation analysis was performed between them. RESULTS: The expression of CD133 was not detected in the control groups while the scores were 7.3 ± 1.4, 5.2 ± 1.1, 2.7 ± 1.0 in junctional zones, tumor cores and edematous areas with immunohistochemistry and 0.79 ± 0.03, 0.38 ± 0.01, 0.20 ± 0.04 with Western blot respectively. There were significant differences between different groups (P < 0.05). Under light microscope, the CD133-positive cells frequently forming perivascular pseudorosettes were dense in junctional zones and mostly clustered near the microvessels in tumor cores and scattered in edematous areas. At a high magnification (200×), the CD34-MVD (/HP) values were 31.3 ± 4.0, 21.8 ± 2.6, 15.3 ± 2.4, 4.7 ± 1.5 respectively in junctional zones, tumor cores, edematous areas and control tissues. Significant differences were also found in these groups (P < 0.05). The expression level of CD133-positive cell was positively correlated with the distribution of CD34-microvessels (r = 0.948, P < 0.05). CONCLUSION: Glioma stem cells tend to assemble in the junctional zones where the microvessels are enriched. There is probably an intimate nourishing relationship with the microvessels. The distribution of glioma stem cells may be related with the invasiveness within glioblastoma.

Expression and Distribution Characteristics of Human Ortholog of Mammalian Enabled (hMena) in Glioma.

OBJECTIVE: To investigate the utility of hMena, a family of enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), we sought to characterize the expression profile and distribution characteristics of hMena in a large panel of glioma samples and determine whether hMena expression levels might correlate with the pathological grade of glioma. METHODS: Sixty-five specimens of glioma with different pathological grades and five control brain tissues were collected. In 6 of the 21 glioblastoma patients, multi-specimens were obtained respectively from the main tumor mass, the junction zone between the tumor and the normal tissue, and adjacent brain tissue 1.5 cm away from the tumor boundary under assistance of neuronavigation system during the operation. Immunohistochemistry was used to detect the expression and distribution characteristics of hMena. hMena expression was analyzed by Western blot in 20 specimens. RESULTS: The hMena expression was negative in control brain tissue but positive in different grades of glioma. The expression rate of hMena was positively correlated with the increasing grade of the World Health Orgnization (WHO) classification (r(s)=0.682, P=0.000). hMena was located in cytoplasm. Positive cells only distributed around the vessels within the tumor mass in low grade glioma, while in high grade glioma, these cells were able to be detected not only in the tumor but also in the boundary zone and adjacent brain parenchyma. In the tumor mass, hMena expressed highly and diffusedly. In the junction zone, hMena positive cells formed radiolitic pattern around the vessels. In adjacent brain parenchyma, single positive cell was scattered. hMena expression was markedly elevated in Grade III and IV glioma compared with Grade II and I. CONCLUSION: Our data suggested that the expression of hMena is closely related to malignant grade of glioma. hMena can label the migrating cells, and indicate the migrating path of glioma cells from the tumor to adjacent tissue along with the vascular basement membranes and tracts of white matter.

[Detection of P-4 and GP-46 expression in Leishmania amazonensis amastigotes and promastigotes by RT-PCR].

OBJECTIVE: To detect the expression level of stage-specific genes in Leishmania promastigotes and amastigotes. METHODS: Total RNAs were isolated from Leishmania amazonensis stationary promastigotes and three sources of amastigotes: freshly obtained from mouse skin lesions, infected J774.G8 macrophages, and transformed from the cultured promastigotes. mRNAs were conversely transcribed into cDNA with SuperScripII reverse transcriptase and oligo dT primers. The polymerase chain reaction (PCR) was used to amplify the specific fragments of amastigote-specific nuclease (P-4) and promastigote-specific membrane glycoprotein (GP-46). PCR products were analyzed in 1.5% agarose gel. RESULTS: A P-4-specific band (273 bp) was observed in all three types of amastigotes with similar density, but it was almost undetectable in promastigotes. In contract, a GP-46-specific band (325 bp) was expressed at a higher level in promastigotes than in all three types of amastigotes. CONCLUSION: Promastigote-derived amastigotes express high level of P-4-specific gene and can be used as a source of amastigotes for biochemical and immunological studies.

Effect of pretreatment with adenosine, diazoxide or ischemic preconditioning on ischemia- reperfusion injury in the limbs of rats.

OBJECTIVE: To study the effect of ischemic preconditioning (PC) and pharmacological preconditioning with adenosine or diazoxide on ischemia-reperfusion (IR) injury in the limbs of rats. METHODS: According to different treatment received before ischemic-reperfusion injury, 66 SD rats were divided into 6 groups including a normal control and a ischemia-reperfusion control group, IP10 group in which the rats received 10-min ischemia followed by 10-min interval for reperfusion for 3 times before IR, IP5 group in which the rats were subjected to 5-min ischemia with 5-min reperfusion intervals for 3 times before IR, adenosine (Ade) pretreatment group and diazoxide (Dia) pretreatment group. Except the normal control group, which consisted of 6 rats, each group contained 12 rats, and IR injury was induced by blocking the blood flow in bilateral limbs for 4 h, followed by reperfusion for 2 or 24 h when twitch and spastic contractility of the tibialis anterior muscle and serum creatine phosphokinase (CPK) were measured. RESULTS: In IP5 and Ade pretreatment group, the twitch tension of the tibialis anterior muscle of the rats was significantly enhanced after 2 and 24 h of reperfusion, achieving the level of the normal control. The twitch tension was also enhanced in rats of IP10 group at 24 h, but at 2 h, though with less effectiveness than that in IP5 and Ade group. Dia pretreatment reduced twitch and tetanic contraction forces of the tibialis anterior muscle after 2 h of reperfusion, but obviously improved twitch tension at 24 h. Improvement in the tetanic tension, however, was seen in none of the groups. Serum CPK of IP5, IP10 and Ade groups after 2 and 24 h while Dia at only 24 h of reperfusion was obviously lower than that of IR group. CONCLUSIONS: Ischemic and Ade preconditioning can protect the limbs of rats from ischemia-reperfusion injury, and Dia has delayed protective effect. IP5 is superior than IP10 and Ade PC can produce similar effect to that of ischemic PC.