Objective To investigate the role of Glycoprotein non-metastatic melanoma protein B(GPNMB)in hypoxia-induced epithelial-mesenchymal transition(EMT)in human chorionic trophoblast HTR-8/SVneo cells.Methods HTR-8/SVneo cells were cultured in vitro to investigate the effect of hypoxia on GPNMB expression.The cells were transfected with either a GPNMB overexpression plasmid(pcDNA3.1-GPNMB),small interfering RNA targeting GPNMB(si-GPNMB-1/2),or their respective negative controls(pcDNA3.1-NC or si-NC),and were also treated with the autophagy agonist rapamycin(Rap).The experimental groups were categorized as follows:Normoxia,Hypoxia,Normoxia/Hypoxia+si-NC or si-GPNMB,Normoxia/Hypoxia+pcDNA3.1-NC or pcDNA3.1-GPNMB,Normoxia/Hypoxia+Rap,and Hypoxia+Rap+pcDNA3.1-NC or pcDNA3.1-GPNMB.GPNMB expression levels were evaluated using qRT-PCR,Western blotting,and immunofluorescence staining.The expression of autophagy-related proteins(LC3B Ⅱ/Ⅰ,p62)and epithelial-mesenchymal transition(EMT)markers(E-cadherin,N-cadherin)was analyzed by Western blotting.Cell migration and invasion capacities were assessed using wound healing and Transwell assays.Results Compared with the Normoxia group,the mRNA and protein levels of GPNMB were downregulated in the Hypoxia group.Additionally,the protein levels of p62 and N-cadherin were reduced,while LC3B Ⅱ/Ⅰ and E-cadherin expression levels were increased(P<0.05).Compared with the Hypoxia+si-NC group,the Hypoxia+si-GPNMB-2 group showed significantly decreased protein levels of p62 and N-cadherin,along with elevated levels of LC3B Ⅱ/Ⅰ and E-cadherin(P<0.05).Compared with the Hypoxia+pcDNA3.1-NC group,the Hypoxia+pcDNA3.1-GPNMB group exhibited opposite trends.Notably,compared with the Hypoxia group,the Hypoxia+Rap group showed increased LC3B Ⅱ/Ⅰ and E-cadherin levels,accompanied by reduced p62 and N-cadherin levels(P<0.05).However,compared with the Hypoxia+pcDNA3.1-GPNMB group,the Hypoxia+Rap+pcDNA3.1-GPNMB group attenuated the promoting effect of GPNMB overexpression on EMT in HTR-8/SVneo cells,as evidenced by decreased p62 and N-cadherin protein expression levels and increased LC3BⅡ/Ⅰ and E-cadherin protein expression levels(P<0.05).Conclusion In hypoxia-induced HTR-8/SVneo cells,GPNMB inhibits autophagy,promotes the epithelial-mesenchymal transition,and enhances cell migration and invasion.

Objective To investigate the causal relationship between abnormal placental lipid metabolism and trophoblast dysfunction in patients with preeclampsia(PE),and to explore the regulatory effects of PPARγ on trophoblast function under hypoxic conditions.Methods Placental tissues were collected from 30 patients with PE and 30 individuals with normal pregnancies at the General Hospital of Ningxia Medical University between October 2020 and November 2021 for the analysis of lipid deposition.A rat model of PE was established,comprising a sham-operated(Sham)group and a reduced uterine perfusion pressure(Rupp)group,with six rats in each group(n=12 total).Human trophoblast cells(HTR-8/SVneo)were cultured in vitro and randomly assigned to four experimental groups:normoxic control,hypoxia,hypoxia+PPARγ agonist(Rosiglitazone),and hypoxia+PPARγ antagonist(T0070907).The expression levels of lipid metabolism-related genes and transcription factors(FASN,FABP4,PPARγ,LXRα)were assessed using RT-qPCR.Western blotting was performed to determine the protein expression levels of PPARγ.Cell migration and invasion capacities were evaluated using scratch wound healing and Transwell assays,respectively.Results Placental lipid deposition in the PE group was significantly higher than that in the control group,particularly in the Rupp model mice(P<0.001).Under hypoxic conditions,the expression levels of FASN and FABP4 were upregulated in trophoblast cells(P<0.001),whereas the expression of PPARγ and LXRα was downregulated(P<0.001).Furthermore,treatment with the PPARγ antagonist T0070907 exacerbated the inhibitory effects of hypoxia on cell function(P<0.001),significantly reducing cell invasion and migration capacity(P<0.001).Additional siRNA-mediated knockdown experiments confirmed that PPARγ deficiency further aggravated hypoxia-induced impairments in cell migration and invasion,and this detrimental effect could not be reversed by Rosiglitazone.Conclusions Abnormal placental lipid metabolism in PE is closely linked to PPARγ-mediated enhancement of lipid synthesis and metabolic dysregulation under hypoxic conditions,which may subsequently impair trophoblast invasion and migration.

Objective To investigate the role of Glycoprotein non-metastatic melanoma protein B(GPNMB)in hypoxia-induced epithelial-mesenchymal transition(EMT)in human chorionic trophoblast HTR-8/SVneo cells.Methods HTR-8/SVneo cells were cultured in vitro to investigate the effect of hypoxia on GPNMB expression.The cells were transfected with either a GPNMB overexpression plasmid(pcDNA3.1-GPNMB),small interfering RNA targeting GPNMB(si-GPNMB-1/2),or their respective negative controls(pcDNA3.1-NC or si-NC),and were also treated with the autophagy agonist rapamycin(Rap).The experimental groups were categorized as follows:Normoxia,Hypoxia,Normoxia/Hypoxia+si-NC or si-GPNMB,Normoxia/Hypoxia+pcDNA3.1-NC or pcDNA3.1-GPNMB,Normoxia/Hypoxia+Rap,and Hypoxia+Rap+pcDNA3.1-NC or pcDNA3.1-GPNMB.GPNMB expression levels were evaluated using qRT-PCR,Western blotting,and immunofluorescence staining.The expression of autophagy-related proteins(LC3B Ⅱ/Ⅰ,p62)and epithelial-mesenchymal transition(EMT)markers(E-cadherin,N-cadherin)was analyzed by Western blotting.Cell migration and invasion capacities were assessed using wound healing and Transwell assays.Results Compared with the Normoxia group,the mRNA and protein levels of GPNMB were downregulated in the Hypoxia group.Additionally,the protein levels of p62 and N-cadherin were reduced,while LC3B Ⅱ/Ⅰ and E-cadherin expression levels were increased(P<0.05).Compared with the Hypoxia+si-NC group,the Hypoxia+si-GPNMB-2 group showed significantly decreased protein levels of p62 and N-cadherin,along with elevated levels of LC3B Ⅱ/Ⅰ and E-cadherin(P<0.05).Compared with the Hypoxia+pcDNA3.1-NC group,the Hypoxia+pcDNA3.1-GPNMB group exhibited opposite trends.Notably,compared with the Hypoxia group,the Hypoxia+Rap group showed increased LC3B Ⅱ/Ⅰ and E-cadherin levels,accompanied by reduced p62 and N-cadherin levels(P<0.05).However,compared with the Hypoxia+pcDNA3.1-GPNMB group,the Hypoxia+Rap+pcDNA3.1-GPNMB group attenuated the promoting effect of GPNMB overexpression on EMT in HTR-8/SVneo cells,as evidenced by decreased p62 and N-cadherin protein expression levels and increased LC3BⅡ/Ⅰ and E-cadherin protein expression levels(P<0.05).Conclusion In hypoxia-induced HTR-8/SVneo cells,GPNMB inhibits autophagy,promotes the epithelial-mesenchymal transition,and enhances cell migration and invasion.

Objective To investigate the causal relationship between abnormal placental lipid metabolism and trophoblast dysfunction in patients with preeclampsia(PE),and to explore the regulatory effects of PPARγ on trophoblast function under hypoxic conditions.Methods Placental tissues were collected from 30 patients with PE and 30 individuals with normal pregnancies at the General Hospital of Ningxia Medical University between October 2020 and November 2021 for the analysis of lipid deposition.A rat model of PE was established,comprising a sham-operated(Sham)group and a reduced uterine perfusion pressure(Rupp)group,with six rats in each group(n=12 total).Human trophoblast cells(HTR-8/SVneo)were cultured in vitro and randomly assigned to four experimental groups:normoxic control,hypoxia,hypoxia+PPARγ agonist(Rosiglitazone),and hypoxia+PPARγ antagonist(T0070907).The expression levels of lipid metabolism-related genes and transcription factors(FASN,FABP4,PPARγ,LXRα)were assessed using RT-qPCR.Western blotting was performed to determine the protein expression levels of PPARγ.Cell migration and invasion capacities were evaluated using scratch wound healing and Transwell assays,respectively.Results Placental lipid deposition in the PE group was significantly higher than that in the control group,particularly in the Rupp model mice(P<0.001).Under hypoxic conditions,the expression levels of FASN and FABP4 were upregulated in trophoblast cells(P<0.001),whereas the expression of PPARγ and LXRα was downregulated(P<0.001).Furthermore,treatment with the PPARγ antagonist T0070907 exacerbated the inhibitory effects of hypoxia on cell function(P<0.001),significantly reducing cell invasion and migration capacity(P<0.001).Additional siRNA-mediated knockdown experiments confirmed that PPARγ deficiency further aggravated hypoxia-induced impairments in cell migration and invasion,and this detrimental effect could not be reversed by Rosiglitazone.Conclusions Abnormal placental lipid metabolism in PE is closely linked to PPARγ-mediated enhancement of lipid synthesis and metabolic dysregulation under hypoxic conditions,which may subsequently impair trophoblast invasion and migration.

Objective To report a newly developed method and procedure to establish a rat model of subarachnoid hemorrhage in detail, and to provide a better model simulating the clinical subarachnoid hemorrhage caused by a ruptured aneurysm for related research.Methods One hundred and twenty healthy SPF 2-3-month old male Sprague-Dawley rats were divided into 4 groups, 30 rats in each group.The three experimental groups were sacrificed at 6, 24 and 72 hours after modeling.Rat models of subarachnoid hemorrhage were established by inserting a fiber core in the internal carotid artery and piercing this artery.Successful establishment of the subarachnoid hemorrhage model was confirmed by observation of breathing, pupil, defecation, urination and inspection at autopsy dissection.The controllability and reproducibility of this model were verified by observation of clinical manifestation and explored by mortality analysis.Results Subarachnoid hemorrhage was successfully induced by fiber core piercing the internal carotid artery at the needed location.Conclusions This method of model preparation is stable and understandable.The operation is nimble, with a good reproducibility.This model can be successfully performed after a short time learning, well simulate the sudden hemorrhage caused by a ruptured aneurysm, and suitable for research on early brain injury and vasospasm after subarachnoid hemorrhage.

In recent years,assisted reproductive technology(ART)is developing rapidly,especially in vitro fertilization and embryo transfer(IVF-ET)technology.The rate of clinical pregnancy in our country is about 40％,and how to improve the rate of clinical pregnancy of IVF-ET has been the focus of the scholars research content.Environmental factors including culture system,temperature,humidity,pH value and volatile organic compounds,all of these can affect the success of IVF-ET.Now the common environmental factors affecting IVF-ET research progress were reviewed as follows.

OBJECTIVETo explore the safety and efficacy of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in preventing chemotherapy-induced neutropenia in patients with breast cancer and non-small cell lung cancer (NSCLC), and to provide the basis for clinical application.

METHODSAccording to the principle of open-label, randomized, parallel-group controlled clinical trial, all patients were randomized by 1∶1∶1 into three groups to receive PEG-rhG-CSF 100 μg/kg, PEG-rhG-CSF 6 mg, or rhG-CSF 5 μg/kg, respectively. The patients with breast cancer received two chemotherapy cycles, and the NSCLC patients received 1-2 cycles of chemotherapy according to their condition. All patients were treated with the combination chemotherapy of TAC (docetaxel+ epirubicin+ cyclophosphamide) or TA (docetaxel+ epirubicin), or the chemotherapy of docetaxel combined with carboplatin, with a 21 day cycle.

RESULTSThe duration of grade 3-4 neutropenia in the PEG-rhG-CSF 100 μg/kg and PEG-rhG-CSF 6 mg groups were similar with that in the rhG-CSF 5 μg/kg group (P>0.05 for all). The incidence rate of grade 3-4 neutropenia in the PEG-rhG-CSF 100 μg/kg group, PEG-rhG-CSF 6 mg group, and G-CSF 5 μg/kg group were 69.7%, 68.4%, and 69.5%, respectively, with a non-significant difference among the three groups (P=0.963). The incidence rate of febrile neutropenia in the PEG-rhG-CSF 100 μg/kg group, PEG-rhG-CSF 6 mg group and G-CSF 5 μg/kg group were 6.1%, 6.4%, and 5.5%, respectively, showing no significant difference among them (P=0.935). The incidence rate of adverse events in the PEG-rhG-CSF 100 μg/kg group, PEG-rhG-CSF 6 mg group and G-CSF 5 μg / kg group were 6.7%, 4.1%, and 5.5%, respectively, showing a non-significant difference among them (P=0.581).

CONCLUSIONSIn patients with breast cancer and non-small cell lung cancer (NSCLC) undergoing TAC/TA chemotherapy, a single 100 μg/kg injection or a single fixed 6 mg dose of PEG-rhG-CSF at 48 hours after chemotherapy show definite therapeutic effect with a low incidence of adverse events and mild adverse reactions. Compared with the continuous daily injection of rhG-CSF 5 μg/kg/d, a single 100 μg/kg injection or a single fixed 6 mg dose of PEG-rhG-CSF has similar effect and is more advantageous in preventing chemotherapy-induced neutropenia.

Antineoplastic Agents ; adverse effects ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; Breast Neoplasms ; drug therapy ; Carboplatin ; administration & dosage ; adverse effects ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; Cyclophosphamide ; administration & dosage ; adverse effects ; Epirubicin ; administration & dosage ; adverse effects ; Female ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Humans ; Incidence ; Induction Chemotherapy ; Lung Neoplasms ; drug therapy ; Neutropenia ; chemically induced ; epidemiology ; prevention & control ; Polyethylene Glycols ; Recombinant Proteins ; administration & dosage ; Taxoids ; administration & dosage ; adverse effects

Objective To explore the effect of extracellular regulated protein kinases (ERK) signaling pathway on early brain injury and autophagy of nerve cell in hippocampus area in rats with subarachnoid hemorrhage (SAH). Methods Forty-eight adult male Sprague-Daw-ley rats were randomly divided into sham group, SAH group, SAH+dimethyl sulfoxide (DMSO) group and SAH+U0126 group, with 12 rats in each group. The SAH model was established with puncture of internal carotid artery. The SAH+U0126 group was injected with U0126 0.05 mg/kg;the sham group and SAH group were injected with normal saline, and the SAH+DMSO group was injected with DMSO 30 min-utes before modeling. They were sacrificed 24 hours after modeling. The brain water content was measured with wet and dry method. The morphology changes of neural cells in hippocampus CA1 were observed by HE staining. The expression of phosphorylation ERK (p-ERK), Beclin-1 and LC3-Ⅱwere detected with immunohistochemical method and Western blotting. Results Compared with the sham group, the brain water content increased (P<0.05), the number of survival neurons decreased (P<0.05), the expression of p-ERK, Beclin-1 and LC3-Ⅱincreased in SAH group (P<0.05). Compared with SAH group, the brain water content increased, the number of survival neurons decreased (P<0.05), the expression of p-ERK, Beclin-1 and LC3-Ⅱ decreased in SAH+U0126 group (P<0.05); and no significant difference was found in SAH+DMSO group (P>0.05). Conclusion The activation of ERK signaling pathway may alleviate early brain injury after SAH by regulation of autophagy.

OBJECTIVETo explore the inhibition effect and mechanism of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP)on myofibroblast differentiation via regulating acetylated tubulin α (Ac-Tub α)in vivo and in vitro.

METHODSSilicotic model were made by SiO2 douched and divided into 6 groups as follows: control (4w, 8w)group, silicotic model (4w, 8w)group and post-or pre-treatment by Ac-SDKP group. Pulmonary fibroblasts were divided into 5 groups: (1) control; (2) Ang II; (3) Ang II+Ac-SDKP; (4) Ang II+Valsartan; (5) Ang II+TCS histone deacetylase (HDAC)6 20b. The localization of Ac-Tub α and α-smooth muscle actin (SMA) were observed by immunohistochemical (IHC) and immunofluorescence staining. The protein levels of Ac-Tub α, α-SMA, collagen type I (col I) and HDAC6 were measured by western blot.

RESULTSIn silicotic nodules and interstitial fibrosis area, positive expression of α-SMA, a classical marker of myofibroblast, was ob-served by IHC, accompanied with absence expression of Ac-Tub α. Furthermore, Ac-SDKP post-treatment could attenuate the levels of col I, α-SMA and HDAC6 to 48.39%, 52.63% and 70.18% compared with the silicotic 8w group respectively. And in Ac-SDKP pre-treatment group, compared with the silicotic 8w group, these protein levels were decreased to 32.26%, 64.91% and 54.39% respectively (P<0.05). The up-regulation of Ac-Tub α was found in Ac-SDKP post-and pre-treatment and increased to 3.00 and 2.90 folds compared with the silicotic 8w group. Compared with control group, the levels of α-SMA, HDAC6 and col I in Ang II group were up-regulated to 1.66, 3.56 and 4.00 folds accompanied with down-regulation of Ac-Tub by 44.44% (P<0.05). Pre-treatment with Valsartan, TCS HDAC6 20b or Ac-SDKP could inhibited all this changes induced by Ang II in vitro.

CONCLUSIONAc-SDKP can inhibit the myofibroblast differentiation and collagen deposition via sup-press HDAC6 and up-regulate the expression of Ac-Tub α in vivo and in vitro.

Actins ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Collagen Type I ; metabolism ; Disease Models, Animal ; Fibroblasts ; cytology ; Lung ; pathology ; Myofibroblasts ; cytology ; drug effects ; Oligopeptides ; pharmacology ; Rats ; Silicon Dioxide ; toxicity ; Silicosis ; drug therapy ; Tubulin ; metabolism

[ ABSTRACT] AIM:To observe the effect of oleanolic acid ( OA) on the expression of Tumor necrosis factor-α( TNF-α) and collagen in silicotic rats in vivo and its possible mechanism.METHODS:Male Wistar rats were divided in-to 4 groups according to the randomized block design:control group, model group, OA group and solvent control group (20 rats in each group) .Except control group, the rats in other groups were induced by intratracheal instillation of silicon di-oxide (SiO2;250 mg/kg).The rats in OA group were intragastrically administered with OA (60 mg/kg) from the second day of giving SiO2 .The rats in solvent control group were gavaged daily with 0.6%sodium carboxymethyl cellulose solution (10 mL/kg).The rats in control group were given normal saline under the same condition for 56 consecutive days.All rats were killed at the 7th, 14th, 28th and 56th days.The lung coefficient was detected and the morphological changes were ob-served.The serum contents of TNF-αwere detected by ELISA.The content of total collagen in the lung tissue was meas-ured.The protein level of nuclear factor-κB ( NF-κB) in the lung tissue was determined by immunohistochemical method. RESULTS:(1) According to the morphological changes, the silicosis model was successfully established.Compared with control group, the lung coefficient and total collagen increased obviously in model group and solvent control group.The lung coefficient and total collagen content in OA group at each time point reduced compared with those in model group and sol-
vent group, and increased compared with those in control group at the corresponding time points.(2) The serum contents of TNF-αin model group and solvent control group significantly increased, peaking at the 14th day, slightly decreasing af-terward, and showing statistically significant difference at each time point compared with those in control group.No signifi-cant difference between model group and solvent group at different time points was observed.OA had inhibitory effect on the contents of TNF-αcompared with model group and solvent group at the corresponding time points.(3) NF-κB in model group and solvent control group significantly increased, peaking at the 28th day, and showing statistically significant differ-ence at each time point compared with those in control group.The NF-κB expression in OA group was similar to model group, but significantly decreased compared with control group at each time point.CONCLUSION: OA inhibits the ex-pression of TNF-αand collagen and attenuates the silicosis fibrosis, which may be related to the NF-κB pathway.