[Effects of Thiomersal on Apoptosis and Autophagy of Leukemia Cell Lines].

OBJECTIVE: To investigate the effects of different concentrations of thiomersal on apoptosis and autophagy regulation of human leukemia cell lines U937, CEM-C1 and BALL-1. METHODS: The inhibitory effect of thiomersal on the proliferation of U937, CEM-C1 and BALL-1 cells was detected by CCK-8 assay. Annexin V-FITC/PI double staining flow cytometry was used to detect the apoptosis rate. Western blot was used to detect the effects of thiomersal on autophagy signaling pathway and the expression of PI3K, Akt, mTOR, p-mTOR, caspase-3 and LC3-II proteins. RESULTS: Within 24 and 48 hours, thiomersal inhibited the proliferation of U937, CEM-C1 and BALL-1 cell lines in a time and dose-dependent manner (r24 h=0.295, r24 h=0.452, r24 h=0.103; r48 h=0.821, r48 h=0.665, r48 h=0.821), but no significant time and dose-dependent effect was observed at 72 hours. After 48 hours treatment of thiomersal, the apoptosis rate of U937, CEM-C1 and BALL-1 cells increased in a dose-dependent manner (r=0.819, r=0.763, r=0.835). After 48 hours treatment of thiomersal, the expression levels of PI3K, Akt, mTOR and p-mTOR protein in U937, CEM-C1 and BALL-1 cells decreased in a concentration-dependent manner, the R value of U937 cells was -0.975, -0.899, -0.925 and -0.915, respectively, that of CEM-C1 cells was -0.960, -0.920, -0.861 and -0.927, and that of BALL-1 cells was -0.939, -0.911, -0.896 and -0.926,. which suggested that thiomersal-induced apoptosis of U937, CEM-C1 and BALL-1 cells might be due to the inhibition of PI3K/Akt/mTOR pathway. Thiomersal promoted the apoptosis of U937, CEM-C1 and BALL-1 cells via caspase-3 pathway, and the expressions of caspase-3 and LC3-II were up-regulated in a dose-dependent manner (r=0.976, r=0.914; r=0.976, r=0.986; r=0.961, r=0.974). CONCLUSIONS: Thiomersal can inhibit the proliferation and promote the apoptosis of U937, CEM-C1 and BALL-1 cells. A certain concentration of thiomersal can down-regulate the expression of PI3K/Akt/mTOR pathway related proteins PI3K, Akt, mTOR and p-mTOR in U937, CEM-C1 and BALL-1 cells, and activate autophagy and apoptosis by down-regulation of PI3K/Akt/mTOR pathway.

Fabrication of attapulgite-based dual responsive composite hydrogel and its efficient adsorption for methyl violet.

In this work, attapulgite (ATP)-based dual sensitive poly (N-isopropylacrylamide-co-acrylic acid) composite hydrogel, P(NIPAM-co-AA)/ATP, was prepared by free radical polymerization. The prepared composite hydrogel was characterized via methods of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), zeta potential analysis and Brunauer, Emmett, and Teller (BET) etc. The composite hydrogel showed pH and temperature sensitive behaviour, with lower critical solution temperature (LCST) of 35°C and highest swelling occurred at pH 8.0. The adsorption of methyl violet (MV) can be controlled by the hydrogel responsiveness, and 95.78% of MV can be removed at pH 8.0 and 35°C. The addition of a small amount of ATP (3 Wt%) can improve the swelling ratio and adsorption capacity. Kinetic analysis demonstrated that the experimental data were best fitted to the pseudo-second order model. Isotherm analysis showed that the equilibrium data followed Langmuir model with the adsorption capacity of 168.35 mg g-1. In addition, the composite hydrogel has high adsorption selectivity for cationic dyes, and MV-loaded hydrogel is easy to regenerate, which can be used for successive adsorption cycles. These results demonstrate that the composite hydrogel has potential application in dye wastewater treatment.

[Relationship of the kallistatin gene expression with male spermatogenic dysfunction and its possible mechanism].

OBJECTIVE: To explore the role of the kallistatin gene in male spermatogenesis and its possible mechanism, and provide some new ideas for the clinical treatment of spermatogenic dysfunction. METHODS: We collected semen samples from the patients with oligospermia (OS) or non-obstructive azoospermia (NOAS) as well as testis tissue from testicular puncture. We detected the differential expression of kallistatin in the seminal plasma and the mRNA and protein expression levels of kallistatin, KLK1, B2R, Bcl-2, casepase-3, Bax and other molecules in the testis tissue, assessed the testicular spermatogenic function using Johnsen's scores, examined the interstitial fibrosis in the testis by Masson and Sirius staining, and analyzed the correlation of the expression level of kallistatin with spermatogenesis, apoptosis and fibrosis. RESULTS: Kallistatin was highly expressed in the seminal plasma and testis tissue. The expression of kallistatin was significantly decreased in the seminal plasma (P < 0.05) and so were those of kallistatin, KLK1 and B2R in the testis tissue of the NOAS and OS patients compared with those in the normal controls (P < 0.01), but no statistically significant difference was found between the expression levels of kallistatin and KLK1 within the same group (P > 0.05). The expression of Bcl-2 in the testis tissue was significantly lower (P < 0.01) and those of Bax and Casepase-3 dramatically higher in the OS and NOAS patients than in the normal males (P < 0.01). Cell apoptosis was negatively correlated with the expression of kallistatin. The results of Masson and Sirius staining showed that the fibrosis of the testis tissue and the ratio of type I/III collagen fibers were markedly increased in the OS and NOAS patients in comparison with the normal controls, even more significantly in the NOAS than in the OS group. CONCLUSION: Decreased expression of kallistatin may be related to spermatogenic dysfunction, and the kallistatin expression plays a regulatory role in the testicular spermatogenesis, probably by regulating cell apoptosis and fibrosis in the testis tissue, but the specific mechanism needs to be confirmed by further studies.

Caspase-11 promotes NLRP3 inflammasome activation via the cleavage of pannexin1 in acute kidney disease.

Ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) in clinic. The activation of NLRP3 inflammasome is associated with inflammation and renal injury in I/R-induced AKI. In the current study we explored the molecular and cellular mechanisms for NLRP3 inflammasome activation following renal I/R. Mice were subjected to I/R renal injury by clamping bilateral renal pedicles. We showed that I/R injury markedly increased caspase-11 expression and the cleavage of pannexin 1 (panx1) in the kidneys accompanied by NLRP3 inflammasome activation evidenced by the activation of caspase-1 and interlukin-1ß (IL-1ß) maturation. In Casp-11-/- mice, I/R-induced panx1 cleavage, NLRP3 inflammasome activation as well as renal functional deterioration and tubular morphological changes were significantly attenuated. In cultured primary tubular cells (PTCs) and NRK-52E cells, hypoxia/reoxygenation (H/R) markedly increased caspase-11 expression, NLRP3 inflammasome activation, IL-1ß maturation and panx1 cleavage. Knockdown of caspase-11 attenuated all those changes; similar effects were observed in PTCs isolated from Casp-11-/- mice. In NRK-52E cells, overexpression of caspase-11 promoted panx1 cleavage; pretreatment with panx1 inhibitor carbenoxolone or knockdown of panx1 significantly attenuated H/R-induced intracellular ATP reduction, extracellular ATP elevation and NLRP3 inflammasome activation without apparent influence on H/R-induced caspase-11 increase; pretreatment with P2X7 receptor inhibitor AZD9056 also attenuated NLRP3 inflammasome activation. The above results demonstrate that the cleavage of panx1 by upregulated caspase-11 is involved in facilitating ATP release and then NLRP3 inflammasome activation in I/R-induced AKI. This study provides new insight into the molecular mechanism of NLRP3 inflammasome activation in AKI.

MIF-mediated NF-κB signaling pathway regulates the pathogenesis of polycystic ovary syndrome in rats.

Polycystic ovary syndrome (PCOS) resulting from abnormal glucose metabolism is a relatively common and complex endocrine disorder among women in their reproductive years, However, the pathogenesis of PCOS is still unclear. The purpose of this study is to investigate the macrophage migration inhibitory factor (MIF) involvement of the nuclear factor (NF)-κB in rats with PCOS. Results indicated that testosterone promoted the increase in the levels of MIF and luteinizing hormone (LH) but inhibited the increase in the level of follicular stimulating hormone (FSH). The MIF antibody could alleviate the process of PCOS to a certain extent. Testosterone promoted the expression of interleukin 1-beta (IL-1ß), interleukin 6 (IL-6), Inducible nitric oxide synthase (iNOS), and tumor necrosis factor alpha (TNF-α); the MIF antibody could reverse this effect. Testosterone could inhibit the expression of NF-κB protein whereas MIF antibody could promote the expression in the ovarian cytoplasm. Testosterone promoted the expression of NF-κB protein in the nucleus, this effect also could be reversed by the MIF antibody. Hyperandrogenism activated the NF-κB pathway. After using the MIF antibody, this effect was reversed. This finding suggested that hyperandrogenism activated the NF-κB pathway through MIF. In short, increased MIF levels activated the NF-κB pathway in ovaries, leading to inflammation and the increase in the levels of relevant inflammatory indicators, which might be one of the important factors in the pathogenesis of PCOS.

Caspase-11/4 and gasdermin D-mediated pyroptosis contributes to podocyte injury in mouse diabetic nephropathy.

Diabetic nephropathy (DN) is characterized by sterile inflammation with continuous injury and loss of renal inherent parenchyma cells. Podocyte is an essential early injury target in DN. The injury and loss of podocytes are closely associated with proteinuria, the early symptom of renal injury in DN. However, the exact mechanism for podocyte injury and death in DN remains ambiguous. In this study we investigated whether pyroptosis, a newly discovered cell death pathway was involved in DN. Diabetic mice were generated by high-fat diet/STZ injections. We showed that the expression levels of caspase-11 and cleavage of gasdermin D (GSDMD-N) in podocytes were significantly elevated, accompanied by reduced expression of podocyte makers nephrin and podocin, loss and fusion in podocyte foot processes, increased inflammatory cytokines NF-κB, IL-1ß, and IL-18, macrophage infiltration, glomerular matrix expansion and increased urinary albumin to creatinine ratio (UACR). All these changes in diabetic mice were blunted by knockout of caspase-11 or GSDMD. Cultured human and mouse podocytes were treated with high glucose (30 mM), which significantly increased the expression levels of caspase-11 or caspase-4 (the homolog of caspase-11 in human), GSDMD-N, NF-κB, IL-1ß, and IL-18, and decreased the expression of nephrin and podocin. Either caspase-4 or GSDMD knockdown by siRNA significantly blunted these changes. In summary, our results demonstrate that caspase-11/4 and GSDMD-mediated pyroptosis is activated and involved in podocyte loss under hyperglycemia condition and the development of DN.

KLF4 initiates sustained YAP activation to promote renal fibrosis in mice after ischemia-reperfusion kidney injury.

Acute renal injury (AKI) causes a long-term risk for progressing into chronic kidney disease (CKD) and interstitial fibrosis. Yes-associated protein (YAP), a key transcriptional cofactor in Hippo signaling pathway, shuttles between the cytoplasm and nucleus, which is required for the renal tubular epithelial cells repair in the acute phase of AKI. In this study we investigated the role of YAP during ischemia-reperfusion (IR)-induced AKI to CKD. Mice were subjected to left kidney IR followed by removal of the right kidney on the day before tissue harvests. Mouse shRNA expression adenovirus (Ad-shYAP or Ad-shKLF4) and mouse KLF4 expression adenovirus (Ad-KLF4) were delivered to mice by intrarenal injection on D7 after IR. We showed that the expression and nucleus distribution of YAP were persistently increased until the end of experiment (D21 after IR). The sustained activation of YAP in post-acute phase of AKI was accompanied by renal dysfunction and interstitial fibrosis. Knockdown of YAP significantly attenuated IR-induced renal dysfunction and decreased the expression of fibrogenic factors TGF-ß and CTGF in the kidney. We showed that the expression of the transcription factor KLF4, lined on the upstream of YAP, was also persistently increased. Knockdown on KLF4 attenuated YAP increase and nuclear translocation as well as renal functional deterioration and interstitial fibrosis in IR mice, whereas KLF4 overexpression caused opposite effects. KLF4 increased the expression of ITCH, and ITCH facilitated YAP nuclear translocation via degrading LATS1. Furthermore, we demonstrated in primary cultured renal tubular cells that KLF4 bound to the promoter region of YAP and positively regulates YAP expression. In biopsy sample from CKD patients, we also observed increased expression and nuclear distribution of YAP. In conclusion, the activation of YAP in the post-acute phase of AKI is implicated in renal functional deterioration and fibrosis although it exhibits beneficial effect in acute phase. Reprogramming factor KLF4 is responsible for the persistent activation of YAP. Blocking the activation of KLF4-YAP pathway might be a way to prevent the transition of AKI into CKD.

[Expression of the kallistatin gene in rat corpus cavernosum].

OBJECTIVE: To investigate the expression of the kallistatin gene in the corpus cavernosum and search for some new molecular targets for the regulation of penile erectile function and treatment of ED. METHODS: Using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), Western blot and immunofluorescence staining, we detected the expression of kallistatin in the rat corpus cavernosum and compared it with that in the aorta. RESULTS: The results of RT-qPCR and Western blot revealed both mRNA and protein expressions of kallistatin in the rat corpus cavernosal tissue, with no statistically significant difference from those in the aorta (P > 0.05). Immunofluorescence staining showed that kallistatin was expressed in both endothelial and smooth muscle cells in the corpus cavernosum and localized in the cytoplasm, with no statistically significant difference from its expression in the aorta (P > 0.05) either. CONCLUSIONS: The kallistatin gene is highly expressed in the corpus cavernosum and localized in cavernosal endothelial and smooth muscle cells, suggestive of its involvement in the cellular function of cavernosal endothelial and smooth muscle cells and its participation in the regulation of penile erectile function.

circRNA-UBAP2 promotes the proliferation and inhibits apoptosis of ovarian cancer though miR-382-5p/PRPF8 axis.

OBJECTIVE: circular RNAs (circRNAs) have been reported to be essential regulators of multiple malignant cancers. However, the functions of circRNAs in ovarian cancer need to be further explored. The aim of our study is to explore the role of circRNA-UBAP2 in ovarian cancer and its mechanism. RESULTS: circRNA-UBAP2 was upregulated in ovarian cancer tissues and cell lines. Knockdown of circRNA-UBAP2 inhibited cell proliferation and promoted cell apoptosis, but circRNA-UBAP2 overexpressed got opposite results. In addition, circRNA-UBAP2 targeted miR-382-5p and downregulated its expression, PRPF8 was a target gene of miR-382-5p. Furthemore, circRNA-UBAP2/miR-382-5p/PRPF8 axis affected the proliferation, apoptosis and cell cycle of ovarian cancer through the mechanism of competing endogenous RNAs (ceRNA). CONCLUSION: circRNA-UBAP2 acted as a ceRNA to sponged miR-382-5p, increased the expression level of PRPF8, and prompted proliferation and inhibited apoptosis in ovarian cancer cells.

Transplantation of adipose-derived stem cells overexpressing inducible nitric oxide synthase ameliorates diabetes mellitus-induced erectile dysfunction in rats.

BACKGROUND: Erectile dysfunction is a major complication of diabetes mellitus. Adipose-derived stem cells (ADSCs) have attracted much attention as a promising tool for the treatment of diabetes mellitus-induced erectile dysfunction (DMED). Inducible nitric oxide synthase (iNOS) plays an important role in protecting penile tissues from fibrosis. The aim of this study was to determine the efficacy of ADSCs overexpressing iNOS on DMED in rats. METHODS: ADSCs were isolated and infected with adenovirus overexpressing iNOS (named as ADSCs-iNOS). The expression of iNOS was detected using western blot analysis and real-time PCR. Rats were randomly assigned into five groups: control group, DMED group, ADSCs group, ADSCs-EGFP group and ADSCs-iNOS group. 5 × 105 cells were given once via the intracorporal route. Two weeks after treatment, erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were obtained and evaluated at histology level. RESULTS: We found that ADSCs-iNOS had significantly higher expression of iNOS at mRNA and protein levels and generated more nitric oxide (NO). ADSCs-iNOS reduced collagen I and collagen IV expression of corpus cavernosum smooth muscle cells (CCSMCs) in cell co-culture model. Transforming growth factor-ß1 expression in CCSMCs reduced following co-culture with ADSCs-iNOS. Injection of ADSCs-iNOS significantly ameliorated DMED in rats and decreased collagen/smooth muscle cell ratio of penile tissues. Moreover, elevated NO and cyclic guanosine monophosphate concentrations were detected in penile tissues of ADSCs-iNOS group. CONCLUSION: Taken together, ADSCs-iNOS significantly improved erectile function of DMED rats. The therapeutic effect may be achieved by increased NO generation and the suppression of collagen I and collagen IV expression in the CCSMCs to decrease penile fibrosis.