[The mechanism of HDAC6 in paraquat-induced autophagy dysfunction of dopaminergic neurons by mediating aggresome-autophagy-lysosomal pathway].

Objective: To explore the mechanism of HDAC6 mediated aggresome-autophagy-lysosome pathway in paraquat-induced autophagy in dopaminergic neurons. Methods: Human neuroblastoma cell (SH-SY5Y cell) was used as model of dopaminergic neurons in vitro. The cells were treated with terminal concentrations of 0, 25, 50, 100, 200 and 400µmol/L PQ for 24 hours, and the cells were induced by 100 µmol/L PQ for different time (0, 12, 24, 36, 48, 60 and 72 h) . Cell viability was detected by CCK-8 assay. The expression levels of HDAC6, α-syn, Dynein IC1/2, LC3, Beclin1, p62 and Lamp-1 were detected by Western blot. Immunofluorescence double-labeling method was used to observe the expression and localization of HDAC6, α-syn, Dynein IC1/2, LC3, Lamp-1 and γ-tubulin in cells. Results: CCK-8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner (R=-0.950、-0.960, P<0.05) .Western blot showed that compared with control group, the protein levels of HDAC6, α-syn, p62 in PQ-exposed group were significantly increased (P<0.05) , but there was a significant decrease in expression level of the ratio of autophagy-related protein LC3 Ⅱ/LC3 Ⅰ, Beclin1, Dynein IC1/2, Lamp-1in PQ-exposed group (P<0.05) . The results of immunofluorescence double-labeling showed that compared with the control group, the fluorescence signals of HDAC6 and α-syn in the PQ-exposed group increased, and the protein expression level increased, while the fluorescence signals of Dynein IC1/2, LC3, and Lamp-1 decreased. The protein expression level is reduced. HDAC6 gradually accumulates from the diffuse shape to the nucleus; Under normal circumstances, α-syn, Dynein IC1/2, γ-tubulin, LC3, and Lamp-1 are mainly distributed in the cytoplasm. After PQ is infected, they gather in the nucleus and co-localize with HDAC6 in the area around the nucleus. Conclusion: PQ may induce abnormal aggregation of α-syn by inducing HDAC6-mediated aggresome-autophagy-lysosomal pathway disorder.

[Autophagic dysfunction contributes to α-synuclein accumulation in dopaminergic neurons induced by paraquat].

Objective: To investigate the effects of Paraquat on autophagy level in SH-SY5Y cell and the mechanism of abnormal aggregation of α-synuclein. Methods: Human neuroblastoma cell (SH-SY5Y cell) was used as model of dopaminergic neurons in vitro. The cells were treated with different concentrations of PQ (0, 18.75, 37.5, 75, 150, 300, 600 µmol/L) for 24 hours, and induced by 150 µmol/L PQ for 0, 12, 24, 36, 48, 60, 72, 96 hours to detect the relative survival rate of cells and determine dose/time-effect relationship. The cells were treated with concentration of 0, 75, 150, 300, 600 µmol/L PQ for 24 hours, and induced by 150 µmol/L PQ for different hours to detect intracellular LDH activity. The expression levels of autophagy-related proteins(LC3I, LC3II, Beclin1 , Vps34 and p62) and α-synuclein were detected by Western blot. The gene expression level of α-synuclein was assayed by Real-time quantitative PCR. The expression level of α-synuclein was also evaluated by immunofluorescence. The cells were pretreated with 100 nmol/L autophagy inducer rapamycin (RAPA) for 6 hours. The expression levels of autophagy-related proteins and α-synuclein were detected by Western blot. Results: CCK8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner; Compared with control group, the activity of LDH in the cell supernatant increased significantly after PQ exposure (P<0.05) ; Western blot analysis showed the ratio of autophagy-related protein LC3II/LC3I, Beclin1 and Vps34 protein expression were significantly lower after PQ treatment while the expression of p62 protein was higher (P<0.05) ; The protein and gene expression of α-synuclein were increased significantly after PQ treatment (P <0.05) ; Immunofluorescence showed the fluorescence intensity of α- synuclein in cells was significantly enhanced (P <0.05) . Compared with PQ group, the expression levels of autophagy-related proteins LC3II/LC3I and Beclin1 were significantly increased whlie α-synuclein protein level was decreased after RAPA induction (P<0.05) . Similarly, the IF result showed the fluorescence signal of α- synuclein significantly decreased after RAPA induction (P<0.05) . Conclusion: Paraquat induced autophagy dysfunction in SH-SY5Y cells, which leads to an abnormal aggregation of α-synuclein.

[Effect of low concentration paraquat on activation of mouse microglia M1/M2 phenotypes].

Objective: To observe the effect of low concentration paraquat (PQ) on activation and phenotypic M1/M2 polarization of mouse microglia cells (BV2) . Methods: BV2 cells were used as model, and cultured in vitro were exposed to paraquat at designed concentrations of 0, 0.015, 0.03, 0.06, 0.12, 0.24, 0.48 µmol/L and 0.05 µmol/L 1-methyl-4-phenylpyridinium (MPP(+)) for 24 h, and cell viability was determined by CCK8 assay. After induced by 0, 0.015, 0.03, 0.06, 0.12 µmol/L PQ and 0.05 µmol/L MPP(+) for 24 h, the contents of tumor necrosis factor-α (TNF-α) , interleukin-6 (IL-6) and IL-1ß in cell culture supernatant were determined by enzyme-linked inmunosorbent assay (ELISA) . Cell migration ability was determined by transwell. Immunofluorescence (IF) and flow cytometry were used to determine the phagocytic capacity of cells. Designed concentrations of 0, 0.03, 0.06, 0.12 µmol/L PQ and 0.05 µmol/L MPP(+) for 24 h, the protein expressions of M1 markers of BV2 (TNF-α, IL-6, IL-1ß, Nitric oxide synthase-iNOS, CD86) and M2 markers of BV2 (Arginase type-1 Arg-1 and Mannose recepteor-CD206) were determined by Western Blot after PQ expourse (0, 0.03, 0.06, 0.12 µmol/L) and 0.05 µmol/L MPP(+) induction. Results: Compared with 0 µmol/L PQ group, proliferation activity of BV2 cells was significantly increased by 0.03~0.12 µmol/L PQ while inhibited by 0.48 µmol/L PQ (P<0.05) . The cell proliferation activity of cells treated with 0.03 µmol/L PQ was significantly increased in 24 hours (P<0.05) . ELISA showed that TNF-α, IL-6 and IL-1ß contents in the cell supernatant of the PQ group were significantly higher than those of 0 µmol/L PQ group, especially in 0.03 and 0.06 µmol/L PQ exposed group (P<0.05) . The results of IF and flow cytometry showed that phagocytic capacity of 0.015, 0.03 and 0.06 µmol/L PQ group was significantly enhanced compared with 0 µmol/L PQ group (P<0.05) . Transwell showed that the cell invasion ability of 0.03, 0.06, 0.12 µmol/L PQ was significantly higher than that of 0 µmol/L PQ group (P<0.05) . Western blot showed that compared with 0 µmol/L PQ group, the expression levels of M1 markers TNF-α, IL-6, IL-1ß, iNOS and CD86 were significantly increased in 0.03 and 0.06 µmol/L PQ exposed group, while the expression levels of M2 markers Arg-1 and CD206 protein were decreased in 0.06 and 0.12 µmol/L PQ exposed group (P<0.05) . Conclusion: Low concentration PQ can abnormally activate BV2 cell, making the transformation of BV2 cell into pro-inflammatory M1 type and inhibiting its transformation into anti-inflammatory M2 type.

[Recent progress in MRI-ultrasound fusion for guidance of targeted prostate biopsy].

Prostate cancer is currently diagnosed by prostate biopsy performed by the transrectal ultrasound-guided technique. However, overdetection of clinical insignificant tumours and missed detection of clinical significant tumours have become problematic. MRI of the prostate, particularly if performed with multiparametric imaging, is capable of detecting clinical significant prostate cancer, which has brought the opportunity to use those images as targets for needle biopsy. Three methods of fusing MRI for targeted biopsy have been recently described: MRI-ultrasound fusion, MRI-MRI fusion ('in-bore' biopsy) and cognitive fusion. Fusion of MRI with ultrasound allows urologists to progress from blind, systematic biopsies to biopsies, which are mapped, targeted and tracked. In the future, MRI-ultrasound fusion for lesion targeting is likely to result in fewer and more accurate prostate biopsies than the present use of systematic biopsies with ultrasound guidance alone.

Renal epithelioid angiomyolipoma without obvious local progress in 10 years: a case report and literature review.

Epithelioid angiomyolipoma (eAMLoma) is an uncommon renal mesenchymal tumor with malignant potential. It is composed of tumor cells arranged in an epithelioid manner. Differential diagnosis from renal cell carcinoma is often challenging because of its epithelioid morphology. Herein is reported a case of eAMLoma, involving a 49-year-old man with eAMLoma. The patient had undergone radical nephrectomy via retroperitoneal laparoscope successfully. He had an uneventful postoperative recovery. The tumor was positive for Desmin, Hmb45, and Sma. We recommend surgical treatment and a follow-up regimen similar to that for renal carcinoma. There was no recurrence and metastases after 1-year follow-up.