BACKGROUND:Steroid-induced femoral head necrosis is mostly caused by long-term and extensive use of hormones,but its specific pathogenesis is not yet clear and needs further study. OBJECTIVE:To screen out the differential miRNAs in osteoclast exosomes after the intervention of Panax notoginseng saponins,and on this basis,to further construct an osteogenic-related ferroptosis regulatory network to explore the potential mechanism and research direction of steroid-induced osteonecrosis of the femoral head. METHODS:MTT assay was used to detect the toxic effects of different concentrations of dexamethasone and different mass concentrations of Panax notoginseng saponins on Raw264.7 cell line.Tartrate resistant acid phosphatase staining and TUNEL assay were used to detect the effects of Panax notoginseng saponins on osteoclast inhibition and apoptosis.Exosomes were extracted from cultured osteoclasts with Panax notoginseng saponins intervention.Exosomes from different groups were sequenced to identify differentially expressed miRNAs.CytoScape 3.9.1 was used to construct and visualize the regulatory network between differentially expressed miRNAs and mRNAs.Candidate mRNAs were screened by GO analysis and KEGG analysis.Finally,the differential genes related to ferroptosis were screened out,and the regulatory network of ferroptosis-related genes was constructed. RESULTS AND CONCLUSION:(1)The concentration of dexamethasone(0.1 μmol/L)and Panax notoginseng saponins(1 736.85 μg/mL)suitable for intervention of Raw264.7 cells was determined by MTT assay.(2)Panax notoginseng saponins had an inhibitory effect on osteoclasts and could promote their apoptosis.(3)Totally 20 differentially expressed miRNAs were identified from osteoclast-derived exosome samples,and 11 differentially expressed miRNAs related to osteogenesis were predicted by target mRNAs.The regulatory networks of 4 up-regulated differentially expressed miRNAs corresponding to 155 down-regulated candidate mRNAs and 7 down-regulated differentially expressed miRNAs corresponding to 238 up-regulated candidate mRNAs were constructed.(4)Twenty-four genes related to ferroptosis were screened out from the differential genes.Finally,12 networks were constructed(miR-98-5p/PTGS2,miR-23b-3p/PTGS2,miR-425-5p/TFRC,miR-133a-3p/TFRC,miR-185-5p/TFRC,miR-23b-3p/NFE2L2,miR-23b-3p/LAMP2,miR-98-5p/LAMP2,miR-182-5p/LAMP2,miR-182-5p/TLR4,miR-23b-3p/ZFP36,and miR-182-5p/ZFP36).These results indicate that Panax notoginseng saponins may regulate osteoblast ferroptosis by regulating the expression of miRNAs derived from osteoclast exosomes,thus providing a new idea for the study of the mechanism of steroid-induced femoral head necrosis.

Objective To investigate the mechanism of action of Ginkgo biloba extract on schizophrenia based on oxidative stress-mediated damage to PV interneurons.Methods 54 SPF grade male mice were selected as experimental subjects,divided into blank group,model group,ginkgo biloba extract 50 mg·kg-1,100 mg·kg-1,150 mg·kg-1 group,and risperidone group.Schizophrenic mouse models were established by intraperitoneal injection of MK-801 0.3 mg·kg-1,and behavioral(open field experiment,Y-maze,forced swimming)tests were conducted.Blood samples and brain tissue were collected 24 h after the last dose,Immunofluorescence staining was used to detect changes in PV neurons in the mouse brain;Detect the content of MAD,GSH Px,and SOD in serum using a reagent kit;ELISA method was used to detect the levels of iron and lipid peroxidation in mouse brain tissue;Western blot was used to detect the protein level of GPX4 in the mouse brain.Results Compared with the model group,the Ginkgo biloba leaf extract 150 mg·kg-1 group and the risperidone group significantly reduced the spontaneous alternation rate of the Y maze and significantly shortened the immobility time of forced swimming(P<0.05);PV neuron staining with varying degrees of enhanced fluorescence intensity;The MDA content in the serum of mice was significantly reduced(P<0.01),while the contents of SOD and GSH px were significantly increased(P<0.05);The iron content in the mouse brain was significantly reduced(P<0.05),the ROS content was significantly reduced(P<0.05),and the GPX4 content in the mouse brain was significantly increased(P<0.05).Conclusion Ginkgo biloba extract has a significant improvement effect on negative symptoms and cognitive impairment in MK-801 induced schizophrenia mouse models,and can also improve PV neuron damage in the prefrontal cortex of schizophrenia mice.Its mechanism may be related to the inhibition of iron death mediated oxidative stress by Ginkgo biloba extract.

Objective To investigate the mechanism of action of Ginkgo biloba extract on schizophrenia based on oxidative stress-mediated damage to PV interneurons.Methods 54 SPF grade male mice were selected as experimental subjects,divided into blank group,model group,ginkgo biloba extract 50 mg·kg-1,100 mg·kg-1,150 mg·kg-1 group,and risperidone group.Schizophrenic mouse models were established by intraperitoneal injection of MK-801 0.3 mg·kg-1,and behavioral(open field experiment,Y-maze,forced swimming)tests were conducted.Blood samples and brain tissue were collected 24 h after the last dose,Immunofluorescence staining was used to detect changes in PV neurons in the mouse brain;Detect the content of MAD,GSH Px,and SOD in serum using a reagent kit;ELISA method was used to detect the levels of iron and lipid peroxidation in mouse brain tissue;Western blot was used to detect the protein level of GPX4 in the mouse brain.Results Compared with the model group,the Ginkgo biloba leaf extract 150 mg·kg-1 group and the risperidone group significantly reduced the spontaneous alternation rate of the Y maze and significantly shortened the immobility time of forced swimming(P<0.05);PV neuron staining with varying degrees of enhanced fluorescence intensity;The MDA content in the serum of mice was significantly reduced(P<0.01),while the contents of SOD and GSH px were significantly increased(P<0.05);The iron content in the mouse brain was significantly reduced(P<0.05),the ROS content was significantly reduced(P<0.05),and the GPX4 content in the mouse brain was significantly increased(P<0.05).Conclusion Ginkgo biloba extract has a significant improvement effect on negative symptoms and cognitive impairment in MK-801 induced schizophrenia mouse models,and can also improve PV neuron damage in the prefrontal cortex of schizophrenia mice.Its mechanism may be related to the inhibition of iron death mediated oxidative stress by Ginkgo biloba extract.

Objective: To investigate the expression and clinical significance of exosomal miR-1231 in plasma of pancreatic cancer (PC) patients and pancreatic cancer cells. Methods: A total of 16 patients who were diagnosed with pancreatic cancer in Hunan Cancer Hospital were collected from April 2016 to August 2017. Meanwhile, 16 healthy volunteers were recruited as the healthy control group at the same period. The plasma exosomes were extracted, and the levels of miR-1231 were detected by qRT-PCR in PC and healthy control groups. Moreover, the clinicopathological significance of exosomal miR-1231 expression was analyzed. Furthermore, the expression of exosomal miR-1231 was detected in several pancreatic cancer cells (MIA PaCa-2, PANC-1, SW1990, AsPC-1 and BxPc-3) and two normal pancreatic epithelial cells (HPDE and human primary pancreatic epithelial cell). Results: qRT-PCR results showed that the expression level of miR-1231 in plasma exosomes of pancreatic cancer patients (1.06±0.46) was significantly lower than that in healthy controls (2.30±0.99; P<0.05). The levels of exosomal miR-1231 in patients with stage Ⅰ-Ⅱ (1.515±0.531), no distant metastasis (1.236±0.461) and no lymph node metastasis (1.337±0.522) were significantly higher than those with stage Ⅲ-Ⅳ (0.848±0.224), distant metastasis (0.757±0.278) and lymph node metastasis (0.838±0.261), respectively (P<0.05 for all). In addition, there were no correlation between exosomal miR-1231 expression and age, sex, smoking history, CA19-9 levels and tumor sites (P>0.05). Furthermore, the expression level of exosomal miR-1231 in pancreatic cancer cell lines (0.142±0.135) was significantly lower than that in normal epithelial cells (1.127±0.179; P<0.05). Conclusions The downregulation of exosomal miR-1231 in plasma of pancreatic cancer patients and pancreatic cancer cells suggests that it is related to the initiation and development of PC. It may be a new diagnostic and prognostic marker for PC.

In this study,RNA interference technique was employed to silence the expression of DNMT1 and/or DNMT3b in human bladder cancer T24 cells.The expression levels of their mRNA and protein were greatly decreased by up to 75% and 65% respectively after T24 cells were transfected with lipofectamine2000 for 72 h,indicating RNA interference is an effective tool in gene knockdown.Proliferation and apoptosis of T24 cells were detected by MTT,and annexin-V-FITC and propidium iodide staining flow cytometry,respectively.It was found that loss of the DNMT1 or DNMT3b expression could inhibit the cell growth and promote the cell apoptosis to some extent.However,combined treatment with shRNA targeting both DNMT1 and DNMT3b mRNA could ob-viously enhance the above effects.It was concluded that simultaneously silencing both genes could result in strong suppressing effect on tumor proliferation and promoting ceil apoptosis than separate use,suggesting combined use of DNMT1 and DNMT3b can achieve a synergistic effect in the CpG island methylation in human bladder tumorigenesis.

Objective To investigate the effect of recombinant plasmid pshRNA-DNMT3b on expression of DNMT3b mRNA and protein and on the proliferation of bladder cancer T24 cells,and research the function of DNMT3b in the process of bladder tumor formation.Methods There were three groups in this study,which are blank controller,HK and pshRNA-DNMT3b(24h,48h,72h),respectively.T24 cells were cultured routinely and transfected by the recombinant plasmids with lipfectamine 2000.The cells were detected by methods of RT-PCR,western blot and MTT.The varying level of DNMT3b mRNA and expression protein,and the conditions of cellular survival rate were observed.Results The recombinant plasmids were successfully transfected into T24 cell lines.The grey valHe of RT-PCR elctrophoretogram was analyzed by the software of Gel-pro analyzer,the rate of blank controller,HK and pshRNA-DNMT3b(24h,48h,72h),was (99.56±1.24)%,(99.12±1.35)%,(75.77±1.42)%,(44.69±1.05)%and(20.52±0.89)%,respectively.The analytical resuit of western blot image was(99.43±1.28)%,(98.90±1.31)%,(67.83±1.02)%,(43.43±1.05)%and(21.92±0.89)%.There was no statistically difference in survival between blank control and HK(P＞0.05).The group of pshRNA-DNMT3b and other two groups had statistical difference only at the 72th hour and the cell inhibitory growth rate only increase 0.45%.Conclusions The recombinant ptasmid pshRNA-DNMT3b can inhibit the expression of mRNA and protein of DNMT3b effectively.However,it has slight function on inhibiting cell proliferation.

OBJECTIVE: The current study was designed to examine the expression of Skp2 gene in laryngeal squamous cell carcinoma (LSCC) and to investigate the role of Skp2 gene in tumorigenesis and progression of LSCC. METHOD: FQ-PCR method was used to examined the expression of Skp2 gene in 40 LSCC and 10 normal laryngeal mucosa tissues, and relationship between its expression and clinical biological factors of patients with LSCC was analyzed. RESULT: The median copy number of Skp2 mRNA expression in LSCC was 6622.54 copy/microg RNA, the median copy number of Skp2 mRNA expression in normal laryngeal mucosa tissues was 0 copy/microg RNA, there was a very significant difference between them (P < 0.01); The positive rate of Skp2 mRNA expression in LSCC and adjacent normal laryngeal tissue were 50%, 0, respectively (P < 0.01). The median copy number of Skp2 RNA expression in LSCC with cervical lymph node metastasis was 617138.4 copy/microg RNA, the median copy number of Skp2 mRNA expression in LSCC without cervical lymph node metastasis was 0 copy/microg RNA, there was a very significant difference between them (P < 0.05); The positive rate of Skp2 mRNA expression in LSCC with and without cervical lymph node metastasis were 100.00%, 35.48%, respectively (P < 0.01). CONCLUSION Skp2 gene might have relation with the cervical lymph node metastasis of LSCC. FQ-PCR is an accurate assay to detecting expression of Skp2 mRNA in patient with LSCC. The level of Skp2 mRNA expression might be a new and more accurate marker, and it can be used to predict cervical lymph node metastasis of LSCC.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; genetics ; Female ; Gene Expression ; Humans ; Laryngeal Neoplasms ; genetics ; Lymphatic Metastasis ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; RNA, Messenger ; genetics ; S-Phase Kinase-Associated Proteins ; genetics ; metabolism