[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.

[Effects of paraquat on the proliferation of human neural stem cells via DNA methylation].

Objective: To investigate the effects of Paraquat on neural stem cell proliferation in vitro and explore the its mechanism based on DNA methylation pathway. Methods: Nestin, ß-tubulin III, and glial fibrillary acidic protein (GFAP) were detected by indirect immunofluorescence assay to evaluate self renewal and differentiation potentia of ReNcell CX human neural stem. The cells were treated with terminal concentrations of 0, 5, 25, 50, and 100µmol/L PQ for 24 hours, and the cells were induced by 50 µmol/L PQ for different time (6, 12, 24, 48 h). Cell viability was determined by MTT assay. The proliferation of neural stem cells was evaluated using Sox2/Brdu and Nestin/Brdu double immunofluorescence staining. The global DNA methylation level was assayed by MethyflashTM methylated DNA Quantification kit. The expression levels of Dnmts mRNA and protein were analyzed by quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western blot, respectively. Results: Immunofluorescence showed that nestin was primarily expressed in proliferative neural stem cell and peotein biomarkers (ß-tubulin III, GFAP) for neuron and astrocyte were expressed in differentiated cells. MTT assay showed PQ induced cell survival rate decrease in a time and dose dependent manner. Double immunfluorescence staining of cells showed colocalization of Sox2 and Brdu. The percentage of Brdu/Sox2 positive cells was significantly lower in the PQ-exposed (25, 50, 100µmol/L PQ treatment) groups compared to control (P<0.05); Meanwhile, The percentage of Brdu/Nestin positive cells was also significantly lower in the PQ-exposed(50,100µmol/L PQ treatment) groups compared to control (P<0.05). The results of global DNA methylation revealed a significant decrease in PQ-exposed groups (P<0.05). Western blot showed that compared with control group, the protein and mRNA levels of Dnmt1, Dnmt3a in PQ-exposed group were significantly decreased (P<0.05), but there was a significant increase in expression level of Dnmt3b in 50, 100 µmol/L PQ-treated group(P<0.05). Conclusion: Paraquat could inhibite the proliferation of human neural stem cells through reducing the level of DNA methylation reaction by suppressing the protein expression and transcription of DNA methylated transferase(Dnmts).

[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.

[The role of the AMPK-mTOR pathway in paraquat-induce autophagy in PC12 cells].

Objective: To investigate the regulation of AMPK-mTOR signal transduction pathway in paraquat-induced autophagy of pheochromocytoma cells (PC12) . Methods: The PC12 cell were treated with terminal concentrations of 0, 25, 50, 100, 200, 300 and 400 µmol/L PQ for 24 hours, and the cells were induced by 300 µmol/L PQ for different time (6, 12, 24, 48 h) . MTT was used to detect the relative survival rate of cells, and the dose/time-effect relationship was determined respectively. The cells were treated with PQ at concentrations of 0, 100, 200 and 300 µmol/L PQ for 24 hours, the lactate dehydrogenase (LDH) activity in the culture supernatant was detected by spectrophotometry. The expression and distribution of autophagic lysosomes were observed by MDC staining. The intracellular reactive oxygen species (ROS) was detected by dichlorofluorescein diacetate (DCFH-DA) . The expression of microtubule-related protein 1 light chain 3 (LC3) was measured by immunofluorescence. The protein level of LC3Ⅱ, p62, Beclin1 and p-AMPK, p-mTOR were detected by Western blot. Results: Compared with the control group, the cell survival rate of the 100, 200, 300, 400 µmol/L PQ group decreased significantly, and showed a dose-dependent pattern (P<0.05) . The survival rate of cells treated with 300 µmol/L PQ decreased significantly with the prolongation of exposure time (12, 24, 48 h) (P<0.05) . Compared with the control group, the activity of LDH in 100, 200, 300 µmol/L PQ-treated group were significantly higher while The fluorescence intensity of ROS was significantly increased (P<0.05) . MDC staining showed the density of autophagic lysosomes and fluorescence intensity in PQ-treated group significantly decreased (P<0.05) . Immunofluorescence results showed the LC3 fluorescence intensity of PQ-treated group decreased which was consistent with MDC staining results. Western blot showed that compared with the control group, the expression levels of autophagy related proteins LC3Ⅱand Beclin1 in PQ-treated group were significantly lower, while the expression level of p62 protein was higher (P<0.05) . p-AMPK protein level decreased and p-mTOR protein expression increased in 200 and 300µmol/L PQ-treatd groups, with statistically significant difference (P<0.05) . Conclusion: AMPK-mTOR signaling pathway played a regulatory role in PQ-induced decreased autophagy of PC12 cell.

[Role of MAPK signaling pathway in epithelial-mesenchymal transition of type II alveolar epithelial cells induced by Paraquat].

Objective: To investigate the roles of p38 mitogen-activated protein kinases (p38 MAPK) , extracellular regulated protein kinases (ERK) and c-Jun N-tenninal kinases (JNK) of MAPK signaling pathway in Paraquat-induced epithelial to mesenchymal transition (EMT) of type II alveolarepithelial cells. Methods: RLE-6NT cells were incubated with different concentrations of PQ (0, 25, 50, 100µmol/L) for 6, 12 and 24 h. Cell morphology alteration was observed under phase-contrast microscopy. Cell viability was determined using an MTT assay. Cell migration ability was detected using scratch wound assay. Protein expression of P-p38 MAP, P-Erk1/2, P-JNK, E-cad, ZO-1, Vimentin and а-SMA were detected by western blot. The level of genes related to fibrosis (COL-I, COL-III, FN and FSP-1) were analyzed via quantitative real-time RT-PCR. Results: Cell morphology started to undergo EMT changes with a phenotype characteristic of mesenchymal cells, including an elongated shape and a lack of tight cell-cell adhesions induced by 100µmol/L PQ treatment in a time-dependent manner. MTT showed that cell viability decreased with increasing PQ concentration (50、100、200、300 µmol/L PQ treatment for 24 h) and increasing treatment time (200 µmol/L PQ treatment for 6, 12, 24, 36, 48 h) . Compared to control group, the expressions of the epithelial phenotype marker E-cad and ZO-1 significantly decreased with PQ treatment (50, 100µmol/L) in a time-dependent manner (P<0.05) . Additionally, the level of the mesenchymal marker (a-SMA, vimentin) dramatically increased with PQ treatment in the same concentration-and time-dependent manner (P<0.05) . Cell migration ability was markedly increased after 24 h of 100 µmol/L PQ treatment compared to control (P<0.05) . The phosphorylated forms of p38 MAPK, Erk1/2, and JNK were increased at 24 h after stimulation with PQ (P<0.05) . This PQ induced (100 µmol/L) phosphorylation was markedly attenuated in the presence of the p38 MAPK, ERK and JNK inhibitors (SB-203580, SP-600125 and PD98059) respectively. Furthermore, RT-PCR showed that PQ significantly induced the upregulation expression of COL I and III mRNA, Fn, and FSP-1 mRNA (P<0.05) . Conclusion: PQ-induced pulmonary fibrosis occurs via EMT, which is mediated by the MAPK pathway.

[Study on the difference of blood coagulation function in patients with traumatic brain injury in plain and plateau area].

Objective: In this study, we tested platelet count (PC), prothrombin time (PT), activated partial thromboplastin time (APTT), and other indicators of coagulation function, and revealed their difference in patients with traumatic brain injury (TBI) between plain and plateau area. Base on the results, we may provide research basis for the therapy of TBI associated coagulopathy in different areas. Methods: 151 TBI patients from Tianjin Medical University General Hospital, and 74 from People's Hospital of Tibet Autonomous Region in the period from Dec 2013 to Dec 2015 were enrolled.Coagulation function, including PC, platelet distribution width (PDW), mean platelet volume (MPV), platelet - large cell ratio (P- LCR), PT, APTT, fibrinogen (FIB), and D- Dimer were tested within 8 h. The difference in patients with TBI between plain and plateau areas were compared and analyzed. Results: Compared with plain area, the PC of patients with TBI in plateau area is lower [(168±49)×109/L vs (196±72)×109/L, P<0.05], while PT and APTT were extended [(13.5±1.3) s vs (12.0±4.0) s, (38±4) s vs(27±6) s, P<0.01]. On the other hand, FIB increases [(3.1±1.2) g/L vs (2.6±1.0) g/L, P<0.01] and D-Dimer decreases [(3.1±3.3) µg/L vs (4.7±3.6) µg/L, P<0.01] in plateau area compared with plain area. Conclusion: Due to the people of plateau area living in hypoxia state, the coagulation function is activated for a long time.Once TBI happens, the platelets and coagulation factors may be excessive consumption, resulting in hypocoagulable state and high risk of rebleeding, while the fibrinolysis system in patients with TBI of plateau area is not activated obviously.Therefore, it should give full consideration to these differences in the treatment of patients with TBI in plateau area, instead of directly copying the standard therapy of the people in plain area.The treatment recommendations should primarily supplement coagulation materials, and antifibrinolytics may unlikely have the therapy effect.

Inhibitory effects of indirubin derivative PHII-7 on invasion and migration in metastatic cancer.

UNLABELLED: PHII-7, a derivative of indirubin, showed significant anti-cancer activities in vivo and in vitro. We asked whether treating human metastatic cancers and multidrug resistant cancer with PHII-7 would inhibit their invasion and migration. Cell growth was tested by MTT assay and colony formation assay. Apoptosis was examined by flow cytometry. Transwell-based assay and wound healing assay were used to examine cell invasion and migration. Real-time PCR assay and western blot assay were performed to test gene expression on mRNA and protein level, respectively. Firstly, we confirmed that MCF-7/ADR cells showed more invasive and migratory properties compared with MCF-7 cells which were associated with several EMT markers, such as E-cadherin, Slug and vimentin. Secondly, we found that slightly toxic doses of PHII-7 decreased the number of cells that invaded a model epithelial basement membrane and that migrated by switching the molecular signature of the cells from mesenchymal to epithelial. And PHII-7 significantly regulated expression of several epithelial-mesenchymal transition (EMT)-related genes, including E-cadherin, Slug, ß-catenin and vimentin. Thirdly, compared with control, PHII-7 inhibited cell proliferation in a time- and dose-dependent manner. Higher doses of PHII-7 also induced apoptosis through activating PARP, caspase-9 and caspase-3. PHII-7 significantly inhibited invasion and migration in both metastatic cancers and multidrug resistant cancer. Our results may provide several data for future application of PHII-7 on drug design and patients treatment. KEYWORDS: PHII-7, invasion, migration, multidrug resistance, epithelial-mesenchymal transition.

[Analysis of clinical phenotype and genotype of Chinese children with disorders of sex development].

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.

[The antibiotic effect of Pseudomonas jinanensis sp. nov].

The Pseudomonas jinanensis has been isolated from soil and screened by antibioticgram assay and spermatogonial assay. It has certain antibiotic properties. The metabolite which has been produced is certainly effective against Staphylococcus aureus and some other positive germs. The preparation of Pseudomonas jinanensis vaccine, namely PJV. It's biological activity on the growth of tumor-bearing mice and the murine spermatogenesis cell. It is considered that enhancement of nonspecific immunologic function of the host might increase the efficacy of praziquantel on schistosomiasis in mice. PJV has certainly produced effectiveness against malignant pleural effusion (effective rate was 76.5%), cancer of lung (effective rate was 58.3%), many solid kinds of cancer (effective rate was 52.1%), and the total effective rate was 64.2%. It could increase the sensitivity of cancer cells to radiation. PJV could stimulate the animal (rabbit) to develop specific antibody. PJV might belong to the class of immunomodulating agents.