Abstract: Objective To establish an immortalized human feeder cell line that efficiently supports the growth of undifferentiated human embryonic stem ( hES) cells. Methods The immortalized TWE3R cell line was established by stably expressing the human E-cadherin gene in the TW2R cell line. The numbers of hESC colonies on TWE3R feeder cells were compared with those on its parental cell lines. After H9 hES cells were cultured on TWE3R cells for 10 consecutive passages, pluripotency marker analysis, embryoid body ( EB) formation assays and teratoma formation assays were conducted. Results We succeeded in establishing the TWE3R human feeder cell line that showed higher efficiency to support the growth of hES cells than its parental cell line. TWE3R feeder cells supported the growth of H9 ESCs at low density ( 5 cells/ cm²). Additionally, after long-term culture on TWE3R feeders, H9 ESCs retained all undifferentiated characteristics, a normal karyotype and the potential to differentiate into derivatives of all three embryonic germ layers. Conclusions The immortalized TWE3R human feeder cell line is capable of supporting efficient growth of human ESCs.

Abstract: Objective To investigate the mechanism of the fibroblast growth factor 21 ( FGF21) / β-klotho / fibroblast growth factor type 1 receptor (FGFR1) pathway in brain injury of type 2 diabetes mellitus (T2DM) model rats with focal cerebral ischemia. Methods SD rats were fed a high fat diet and injected intraperitoneally with streptozotocin (STZ) to establish the T2DM rat model. The middle cerebral artery occlusion ( MCAO) model was established by the suture method. Rats were divided into normal control group ( Control group), simple cerebral ischemia group ( MCAO group), T2DM cerebral ischemia group (T2DM + MCAO group), T2DM cerebral ischemia FGFR1 inhibitor PD173074 treatment group ( PD173074 group ) and T2DM cerebral ischemia FGFR1 agonist PF05231023 treatment group (PF05231023 group) with 12 rats in each group. PD173074 and PF05231023 groups were injected with 5 mg / kg PD173074 and 5 mg / kg PF05231023 respectively at 30 minutes before MCAO. After MCAO for 24 hours, the neurological deficit score was calculated and the rats were sacrificed to obtain brain tissue samples. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to detect neuronal apoptosis. Real-time quantitative PCR was used to detect mRNA expression of FGF21, β-klotho and FGFR1. Protein expression of FGF21, β-klotho, FGFR1, platelet endothelial cell adhesion molecule (CD31), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) was detected by Western blot. Results Compared with the control group, the neurological deficit score, apoptosis rate of nerve cells, mRNA and protein levels of FGF21, β-klotho, FGFR1 and protein levels CD31, ET-1, VEGF were significantly increased in MCAO and T2DM + MCAO groups (P<0. 05). Compared with the T2DM + MCAO group, the neurological deficit score, apoptosis rate of nerve cells, protein levels of CD31, ET-1 and VEGF were increased in the PD173074 group, while the mRNA and protein levels of FGF21, β-klotho and FGFR1 were decreased (P< 0. 05). Moreover, the neurological deficit score, apoptosis rate of nerve cells and the protein levels of CD31, ET-1 and VEGF in the PF05231023 group were decreased, while the mRNA and protein levels of FGF21, β-klotho and FGFR1 were increased (P< 0. 05). Conclusions Activation of the FGF21 / β-klotho / FGFR1 pathway may play an important protective role in focal cerebral ischemia model rats with T2DM.

Abstract: Objective To investigate the difference between BALB/ c mutant curly mice and normal BALB/ c mice in the establishment of tumor models by hepatic portal vein injection and establish a quantitative analysis method of images using Image J software. Methods H₂₂ cells were injected into the liver through the portal vein of the two groups and livers were removed 21 days later. The largest diffuse lesion of the liver lobe was selected for analysis. The vertical view of the tumor appearance was obtained by acquiring images, and then Image J software was used for quantitative analysis and comparison of liver tumor images. Results Multiple diffuse tumors were found in both groups. The multiple and diffuse degree of tumors in the curly mice group was significantly higher than that in the normal mice group, and the tumors in curly mice group were more closely related to the surrounding normal liver tissue infiltration than that in normal mice group. The result of Image J software analysis showed that there was no significant difference between the two groups, but the proportions of tumors and tumors in the liver of curly mice were significantly higher than those in normal mice (P<0.01). Conclusions The animal model of advanced liver tumors was established successfully. The ability of BALB/ c mutant curly mice to model tumor cells via portal vein injection was significantly better than that of normal BALB/ c mice. Moreover, a quantitative analysis method of images was established using Image J software.

Abstract: Objective To investigate the effect and mechanism of androgens on mitochondrial functions of gastric mucosa in aged gonadectomized male (GDX) rats. Methods 36 male SD rats were randomly divided into three groups: sham operation group (Sham, n= 12), model group (GDX, n= 12) and androgen-supplemented model rats ( n= 12). GDX rats were used for modeling. After modeling, the AG group was administered testosterone ( 13. 6 mg / ( kg·d), dissolved in sesame seed oil). Rats in sham operation and model groups were treated with sesame seed oil. After the treatment, gastric tissue samples were collected and the levels of Mn-SOD, GSH-Px, GSH, GSSG, GSH/ GSSG and ROS in gastric mucosa mitochondria were measured by kits, the mitochondrial membrane potential was measured by rhodamine 123 fluorescence, and apoptosis was analyzed by flow cytometry. In vitro, GES-1 cells were used as the research object. H₂O₂ was used to induce cellular injury. The effects of androgens and ALDH2 inhibitor disulfiram on cell viability and apoptosis were investigated. Results Compared with Sham group, ROS and GSSG levels, gastric mucosal cell apoptosis and cleaved Caspase-3 and cytoplasmic Cyt c protein expression were significantly increased in the GDX group (P<0. 05), while Mn-SOD, GSH-PX, GSH and GSH/ GSSG levels, the mitochondrial membrane potential, ATP level and mitochondrial Cyt c and ALDH2 protein expression were decreased significantly (P<0. 05). After androgen treatment, the above changes in GDX elderly rats were reversed significantly ( P< 0. 05). In vitro, androgen treatment significantly reversed damage induced by H₂O₂ in GES-1 cells (P<0. 05). However, when combined with Disulfiram, the therapeutic activity of androgen was reduced significantly ( P< 0. 05). Conclusions Androgen deficiency leads to mitochondrial dysfunction and increased mitochondrial ROS accumulation in gastric mucosal cells of elderly rats and induces gastric mucosal cell apoptosis, which is at least partly related to inhibition of the antioxidant function of ALDH2.

Abstract: Objective To investigate the effect of miR-27a on Streptococcus pneumoniae ( SP) -induced injury of HPAEpiCs by regulating autophagy mediated by the PI3K/ AKT / mTOR pathway. Methods HPAEpiCs were infected with 1× 10⁸ CFU/ mL SP. At 48 hours before induction the cells were transfected with miR-27a-NC ( miR-27a-NC group) , miR-27a-inhibitor ( miR-27a-inhibitor group) , pcDNA-NC ( pcDNA-NC group) or pcDNA-PI3K ( pcDNA- PI3K group) or cotransfected with miR-27a-NC and Si-NC (miR-27a-NC+Si-NC group) , miR-27a inhibitor and Si-NC (miR-27a inhibitor+Si-NC group) , miR-27a-NC and Si-PI3K (miR-27a-NC+Si-PI3K group) , or miR-27a-inhibitor and Si-PI3K (miR-27a-inhibitor+Si-PI3K group) using a Lipofectamine 3000 transfection kit. Non-induced cells were used as the control group, and induced, but untransfected, cells were used as the induction group. qRT-PCR was used to measure the expression level of miR-27a in normal and SP-induced alveolar epithelial cells. Bioinformatics prediction and dual luciferase assays were used to verify the targeting relationship between miR-27a and PI3K. CCK-8 Assays was used to assess proliferation activity. Flow cytometry was used to measure the apoptosis rate. ELISA were used to detect the contents of IL-6 and IL-10 in culture supernatants. Western blot was used to measure the expression levels of PI3K and Beclin1, the LC3-II/ I ratio and the phosphorylation levels of AKT and mTOR. Results Compared with the control group, the expression level of miR-27a, apoptosis rate, IL-6 content, Beclin1 protein expression level and LC3-II/ I ratio were higher in the induction group (P<0. 05) and the PI3K protein expression level, cell proliferation rate, IL-10 content and phosphorylation levels of AKT and mTOR were lower (P<0. 05) . Compared with the miR-27a-NC group, the expression level of miR-27a, apoptosis rate, IL-6, Beclin1 protein expression level and LC3-II/ I ratio were lower in the miR-27a-inhibitor group content ( P< 0. 05) , the cell proliferation rate, content of IL-10, the protein phosphorylation levels of AKT and mTOR were higher ( P< 0. 05) . Compared with the pcDNA-NC group, the apoptosis rate, IL-6 content, Beclin1 protein expression level and LC3-II/ I ratio were lower in the pcDNA-PI3K group ( P< 0. 05) and the PI3K protein expression level, cell proliferation rate, IL-10 content and phosphorylation levels of AKT and mTOR were higher (P<0. 05) . Conclusions During injury of HPAEpiCs induced by SP, miR-27a inhibits the PI3K/ AKT / mTOR signaling pathway and promotes autophagy. Inhibition of miR-27a activates the PI3K/ AKT / mTOR signaling pathway, inhibits autophagy and alleviates SP-induced injury of HPAEpiCs.

Abstract: Objective To explore the relationship between expression of matrix metalloprotein-9 (MMP-9) and tissue inhibitor of metalloproteinases 1 ( TIMP-1) in premature infants with FIRS and bronchopulmonary dysplasia. Methods This study was designed with clinical and animal experiments. Clinical experiments: From May 2018 to December 2019, 118 preterm samples were obtained and divided into control group and experimental group, to observe inflammatory indicators (IL-6, MMP-9, TIMP-1 and M/ T ratio) and pathological tissue infiltration by inflammatory cells and analyze the relationship between inflammatory indicators of the groups. Animal experiments: Establish the pregnant mice and randomly divided them into three groups, lipopolysaccharide group (LPS, n= 10), retinoic acid group (RA, n= 10) and normal saline group (NS, n= 10). The inflammatory indexes of lung tissues and inflammatory cell infiltration in placental pathological tissues in the three groups were assessed, and the relationship between the inflammatory indexes of the three groups was analyzed. Results The expression levels of IL-6, MMP-9, TIMP-1 and the M/ T ratio in cord blood of premature infants in the FIRS group of different gestational ages were significantly higher than those in the control group (P<0. 01). The incidence of BPD in the FIRS group was higher than that in the control group at the same gestational age. Animal experiments: The expression levels of MMP-9 and TIMP-1 and the M/ T ratio in newborn (fetal) rats of different FIRS age groups were significantly higher than those of the control group at the same age (P<0. 01). After RA treatment, the inflammatory index was higher than that in the control group at the same age (P<0. 01), but lower than that in the FIRS group at the same age (P<0. 01). IL-6 was positively correlated with MMP-9, TIMP-1 and the M/ T ratio in mice lung tissue. Conclusions FIRS regulates MMP-9, TIMP-1 levels and the M/ T ratio, mediates BPD and the application of an MMP-9 inhibitor plays a protective role in the process of lung injury, which provides a potential drug target for clinical treatment.

Abstract: Objective To investigate the protective effect of etomidate (Eto) mediated via the Toll-like receptor 4 (TLR4) pathway on acute lung injury in septic shock rats. Methods 75 rats were randomly divided into sham operation, model, model-lipopolysaccharide (LPS), model-Eto and model-Eto-LPS group with 15 rats in each group. Except for the sham operation group, the other groups underwent cecal ligation and perforation to establish the septic shock model. The sham operation group only underwent a laparotomy. 30 minutes before surgery, the model-LPS group was intraperitoneally injected with 15 mg / kg LPS, the model-Eto group was intraperitoneally injected with 60 mg / kg etomidate, and the model- Eto-LPS group was intraperitoneally injected with 60 mg / kg etomidate and 15 mg / kg LPS, model and sham operation groups were intraperitoneally injected with saline. The level of serum endotoxin (ET) and the levels of IL-1β, IL-6 and TNF-α in BALF were measured at 5 hours after the operation. Pathological changes were observed in lung tissue. mRNA and protein expression of TLR4, myeloid differentiation factor (MyD88), and nuclear factor-κB p65 (NF-κB p65) was detected in lung tissue. Results Compared with the sham operation group, ET contents and IL-1β, IL-6 and TNF-α levels in BALF were higher in the model, model-LPS, model-Eto and model-Eto-LPS groups, in the order of model-Eto grouP< model-Eto-LPS grouP<model grouP<model-LPS group (P<0. 05). HE staining showed that alveoli of model and model-LPS were obviously congested, the alveolar wall and lung interstitium had thickened, and a large number of inflammatory cells had infiltrated. The model-LPS group had more severe lesions. In model-Eto and model-Eto-LPS groups, the lesions were relieved with occasional alveolar capillary congestion, and inflammatory cell infiltration. The reductions were more obvious in the model-Eto group. Compared with the sham operation group, the relative expression of TLR4, MyD88 and NF-κB p65 mRNA and protein in lung tissues were higher in model, model-LPS, model-Eto and model-Eto-LPS groups in the order for model-Eto grouP<model-Eto-LPS grouP<model grouP<model-LPS group (P<0. 05). Conclusions Eto has a protective effect on acute lung injury in septic shock rats, which may play a regulatory role by inhibiting the TLR4 pathway.

Abstract: Objective To explore the mechanism of miR-214 in immunoregulation of type II alveolar cells infected by Mycobacterium tuberculosis. Methods A549 cells were infected with BCG and H37Rv. qRT-PCR was used to measure the expression levels of miR-214 and FGF9. Western blot was used to measure the expression level of FGF9. SP- A, SP-D, TLR2, TLR4, IL-10, TNF-α, TNF-γ, IL-1β and IL-8 were measure by ELISA. Western blot was used to detect expression of PI3K, AKT and p-AKT. Results Compared with those in cells infected with BCG, expression of miR-214 was increased, and expression of FGF9 was decreased in A549 cells infected with H37Rv. Compared with those in the NC- mimic+oe-NC group, SP-A, SP-D and IL-10 were decreased significantly, TLR2, TLR4, TNF-α, TNF-γ, IL-1β and IL-8 were increased significantly. PI3K and AKT/ p-AKT expression was increased significantly in the miR-214-mimic+oe-NC group, SP-A, SP-D and IL-10 were increased significantly. TLR2, TLR4, TNFα, TNF-γ, IL-1 β and IL-8 were decreased significantly, PI3K and AKT/ p-AKT expression was decreased significantly in the NC mimic+oe-FGF9 group. Compared with those in the NC-mimic+oe-NC group, SP-A, SP-D and IL-10 were increased significantly, TLR2, TLR4, TNF-α, TNF-γ, IL-1 β and IL-8 were decreased significantly. PI3K and AKT/ p-AKT expression was decreased significantly in the miR-214 mimic+oe-FGF9 group. Conclusions miR-214 inhibits FGF9 expression and activates the PI3K/ AKT signaling pathway, which leads to the excessive immune response of type II alveolar cells infected by Mtb and the development of inflammation.

Abstract: Objective To investigate the effect of Lycium barbarum polysaccharide and UCA1 siRNA individually, or in combination, on the proliferation and apoptosis of SH-SY5Y cells induced by MPP⁺ and its mechanism. Methods SH-SY5Y cells were treated with MPP⁺(0. 25, 0. 5, 1 and 2 mmol / L for 24 hours) and LBP (50, 100, 200 and 400 μg / mL Lycium barbarum polysaccharide pretreatment for 1 hour and then treated with MPP⁺ for 24 hours). The optimal concentrations of MPP⁺ ( 1 mmol / L) and LBP ( 400 μg / mL) were selected for subsequent experiments. The experiment was divided into four groups: control group ( without treatment ), MPP⁺ group ( cells were treated with 1 mmol / L MPP⁺ for 24 hours), Lycium barbarum polysaccharide group ( cells were pretreated with 400 μg / mL Lycium barbarum polysaccharide for 1 hour and then treated with 1 mmol / L MPP⁺ for 24 hours), si-UCA1 group ( cells were treated with 1 mmol / L MPP⁺ and then transfected with si-UCA1) and barbarum polysaccharide + si-UCA1 group. MTT assays were used to assess cell viability and flow cytometry was used to measure the apoptosis rate, ROS level and membrane potential. Results Compared with the control group, the cell survival rate and membrane potential in the MPP⁺ group were reduced significantly and the apoptosis rate and ROS level were increased significantly (P<0. 05). Compared with the MPP⁺ group, the cell survival rate and membrane potential in Lycium barbarum polysaccharide and si-UCA1 groups were increased significantly and the apoptosis rate and ROS level were decreased significantly (P<0. 05). Compared with Lycium barbarum polysaccharide and si-UCA1 groups, the cell survival rate and membrane potential in the LBP+si-UCA1 group were increased significantly and the apoptosis rate and ROS level were decreased significantly ( P< 0. 05 ). Conclusions Lycium barbarum polysaccharide and inhibition of UCA1 expression promote the proliferation of SH-SY5Y cells induced by MPP⁺ and inhibit apoptosis. Their combination has a more obvious effect on cell proliferation and apoptosis. The mechanism may be related to inhibition of mitochondrial apoptosis.

Abstract: Objective To explore the effect of honokiol on serum inflammatory factors, extracellular matrix component expression, and apoptotic pathway-related protein expression in rats with D-galactose-induced intervertebral disc degeneration. Methods Forty-five SD rats were randomly divided into control ( Control), D-galactose ( D-gal) model (Model), honokiol low dose (2 mg / kg), honokiol medium dose (4 mg / kg) and honokiol high dose (8 mg / kg) groups, with 9 rats in each group. The rat model of intervertebral disc degeneration was established by subcutaneous injection of D- galactose in model and honokiol groups, and the Control group was subjected to subcutaneous injection of normal saline. After successful modeling, administration group rats were gavaged with magnolol at 2, 4 and 8 mg / kg, and the remaining rats were gavaged with normal saline in each group. Rats were sacrificed after 4 weeks of continuous administration. HE and safranin O staining was used to observe and compare the morphology and histology of intervertebral discs in each group. The mRNA levels of extracellular matrix components were measured by qRT-PCR in rat intervertebral discs. Changes of interleukin IL-1β, IL-6, tumor necrosis factor-α ( TNF-α) and IL-10 in rat serum were detected by ELISAs. TUNEL staining was performed to observe apoptosis of rat intervertebral disc. Protein expression of B-cell lymphoma protein 2(Bcl- 2) / Bcl-2 associated X protein ( Bax), Caspase-9, Caspase-3 and c-Myc was detected by Western blot. Results The scores of intervertebral disc histomorphology in the D-galactose group and honokiol low and medium dose groups were significantly higher than that in the control group (P<0.05). Compared with that in the D-galactose group, the scores of intervertebral disc histomorphology in medium and high dose honokiol groups were significantly lower (P<0.05). Cartilage damage in the model group and the honokiol low dose group was severe, and that in honokiol medium and high dose groups was slightly damaged. Honokiol significantly increased the expression levels of SRY-related high mobility group-box gene 9 ( SOX-9), collagen II and aggrecan (P<0.05). The levels of IL-1β, IL-6, TNF-α and IL-10 in the D-galactose group and honokiol medium and high dose groups were significantly higher than those in the control group (P<0.05). Compared with that in the D-galactose group, the levels of IL-1β, IL-6 and TNF-α in the honokiol medium and high dose groups were reduced significantly (P<0.05), and the content of IL-10 was increased significantly (P<0. 05). Compared with that in the control group, the number of apoptotic cells in the model group and honokiol low and medium dose groups was increased significantly (P< 0. 05), the Bcl-2/ Bax ratio decreased significantly (P< 0.05), and the protein levels of Caspase-9, Caspase-3 and c-Myc were increased significantly ( P< 0.05). Compared with that in the model group, the number of apoptotic cells in the honokiol medium and high dose groups was decreased significantly (P<0. 05), the Bcl-2/ Bax ratio was increased significantly (P<0.05), and the protein levels of Caspase-9, Caspase-3 and c-Myc were decreased significantly (P<0.05). Conclusions Honokiol has obvious therapeutic effects on lumbar intervertebral disc degeneration induced by D-galactose.

Abstract: Objective To explore the effect of a long-term high protein diet on liver and adipose tissues of ovariectomy (OVX) mice and the role of glucagon-like peptide-1 (GLP-1). Methods 32 8-week-old C57BL/ 6 female mice were randomly divided into control (Control), model (OVX), high protein (OVX+HP) and low protein (OVX+LP) groups with eight rats in each group. After ovariectomied, OVX, OVX + HP and OVX + LP groups were provided with standard, high protein and low protein diets, respectively. The control group received the same operation to preserve the ovaries and were provided with a standard diet. The mice were weighed weekly and sacrificed at the end of 24^th week. The liver, colon and retroperitoneal adipose tissue were collected, and retroperitoneal adipose tissue was weighed. HE staining was used to observe pathological changes of the liver and retroperitoneal adipose tissue. An enzyme-linked immunosorbent assay was used to measure serum GLP-1 levels. Real-time quantitative PCR was used to measure the expression of sterol regulatory element-binding protein-1c (SREBP-1c) in the liver and GLP-1 in colon tissues. Immunohistochemical staining was used to detect expression of SREBP-1c protein in the liver. Results At the end of 24^th week, compared with the control group, the OVX group gained weight (P<0. 05) and retroperitoneal adipose tissue was increased (P<0. 05). A large number of lipid droplets in hepatocytes and the retroperitoneal adipocyte volume were increased in the OVX group. GLP-1 mRNA expression in colon tissues of the OVX group was decreased (P<0. 05), and the serum GLP-1 content in the OVX group was also decreased (P<0. 05). SREBP-1c mRNA and protein expression in liver tissues was increased in the OVX group (P<0. 05). Compared with the OVX group, the OVX+HP group showed a decrease in body weight (P<0. 01) and a significant decrease in retroperitoneal adipose tissue (P<0. 001). A small amount of lipid droplets was observed in hepatocytes, and retroperitoneal adipose cells had shrunk in pathological sections of the OVX+HP group. GLP-1 mRNA expression in colon tissues was increased in the OVX + HP group ( P< 0.01). Serum GLP-1 content was significantly increased in the OVX+HP group (P<0. 001). SREBP-1c mRNA and protein expression in liver was decreased in the OVX +HP group ( P < 0. 001). There was no statistically significant difference in weight, retroperitoneal fat and the mRNA expression of GLP-1 and SREBP-1c between OVX+ LP and OVX groups. Conclusions A long-term high protein diet improved weight gain and liver steatosis in OVX mice, which may be related to the high protein diet promoting GLP-1 secretion in intestines and downregulating the protein expression of SREBP-1c in the liver, which plays a similar role to estrogen.

Abstract: Objective To use the SYRCLE animal experiment risk assessment tool, ARRIVE 2019 guidelines and GSPC checklist to evaluate the report quality of animal experiments of electroacupuncture in the neurogenic bladder after spinal cord injury and determine method to improve the quality of animal experiment reports. Methods CNKI, WANFANG, VIP, SinoMed, PubMed, Web of science and Cochrane library databases were searched to find animal experiments of electroacupuncture in the neurogenic bladder in accordance with the SYRCLE animal experiment risk assessment tool, ARRIVE 2019 guidelines and GSPC list. These data were extracted from each database and statistical analysis was performed using Excel 2019. Stata software was used for systematic evaluation and analysis. Results Twenty- six studies were finally included. The total scores of all studies in the SYRCLE animal experiment risk assessment tool were generally low, which ranged from 10 to 14 points and the risk of bias was high. Only 18 studies used the “random number table method ”. No studies used blinded method in animal allocation or result measurement. There were 21 studies in which the number of animals was missing during the experiment, but none of them took measures or described the effect on experimental result. The low-risk rate of essential items in the ARRIVE 2019 guideline is 43. 14%, while the low-risk rate of recommended items is only 23. 56% and the low-risk rate of the GSPC list is 20. 45%. There were 21 studies that reported animal exclusion mostly due to death, other complications, or model failure. Eighteen studies used the random number table method and eight studies only mentioned randomness without specifying the method. Eight studies described the animal species, age, sex and weight in detail. Two studies both provided animal license numbers and indicated that they had passed ethical review. Three studies described measures to reduce pain and suffering during the experiments. Six studies discussed the limitations of the research. Nine studies described the current research progress of NGB after SCI and provided relevant references, indicated the deficiencies in current treatments and established clear research goals. Two studies blindly described specific outcome indicators and measurement method to experimental staff. The 26 studies discussed the main findings and explained the clinical significance of the result and the significance of the entire science. Systematic evaluation of the study result showed that electroacupuncture improved the maximum bladder volume and bladder compliance and reduced the bladder leakage point pressure compared with the model group. Conclusions The current publicly published animal studies on electroacupuncture of the neurogenic bladder after spinal cord injury are generally of low quality and descriptions of multiple items are incomplete, which prevents readers from Objectively and accurately assessing the level of risk of bias that the animal experiment may produce and even affects the reader’ s understanding of animal research. Judgments can be further translated into clinical research. It is recommended to take targeted measures to further promote and use SYRCLE animal experiment bias risk assessment tools and experimental research report specifications (ARRIVE 2019 guidelines and GSPC list),which can further improve the design, implementation, and reporting of animal experiments and improve the reproducibility of animal experimental research and result.

Abstract: Objective To investigate the effect and mechanism of OSTN-AS1 on the proliferation, migration, and invasion of prostate cancer cell PC-3M. Methods The cancerous tissues and corresponding adjacent tissues of 25 prostate cancer patients were collected, and then RT-qPCR was used to measure the expressionof OSTN-AS1 in the tissues. PC-3M cells were transfected with OSTN-AS1 small interfering RNA and / or miR-330 inhibitor. Flow cytometry was used to analyze the cell cycle. A colony formation assay was used to analyze the number of cell clones. Transwell assays were used to analyze the migration and invasion. RT-qPCR was used to measure the expression of miR-330 and MMP-2 mRNA. Dual luciferase reporter gene assays were used to verify the regulatory relationships between OSTN-AS1 and miR-330 as well as miR-330 and MMP-2. Transfected PC-3M cells were injected into nude mice subcutaneously. After 4 weeks, the tumors were harvested and weighed. Results Expression of OSTN-AS1 was increased in prostate cancer tissue (P<0.05), but expression of miR-330 was decreased (P<0.05). Down-regulating OSTN-AS1 blocked cell cycle progression of PC-3M cells and inhibited cell proliferation, migration, and invasion in vitro. At the same time, down-regulating OSTN-AS1inhibited tumor growth in vivo. Down-regulating miR-330 promoted cell cycle progression, proliferation, migration and invasion of PC-3M cells in vitro. At the same time, down-regulating miR-330 promoted tumor growth in vivo. OSTN-AS1 negatively regulated miR-330 expressionand miR-330 negatively regulated MMP-2 expression. The inhibitory effects of down-regulating OSTN-AS1 on the cell cycle, proliferation, migration and invasion of PC-3M cells in vitro and tumor growth in vivo could be reversed by down-regulating miR-330. Conclusions OSTN-AS1 may promote the development of prostate cancer by regulating the miR-330 / MMP-2 axis, which may a molecular target for prostate cancer treatment.

Abstract: Objective To explore the effect of lncRNA HOXA-AS3 on cisplatin resistance in small cell lung cancer cells and its regulatory effect on miR-340-5p. Methods The expression levels of HOXA-AS3 and miR-340-5p in lung cancer tissues and adjacent tissues were measured by qRT-PCR. Small cell lung cancer cell line DMS114 was cultured in vitro and drug-resistant cell line DMS114 / DDP was established by increasing the concentration of cisplatin. si-NC, si- HOXA-AS3, si-HOXA-AS3, anti-miR-NC, si-HOXA-AS3 and anti-miR-340-5p were transfected into DMS114 / DDP cells. The expression levels of HOXA-AS3 and miR-340-5p in cells were measured by qRT-PCR. CCK-8 assays were used to measure cell survival rate and calculate the IC₅₀ value of cisplatin. Flow cytometry was used to assess the apoptosis rate. Dual luciferase reporter assays were used to analyze the targeting relationship between HOXA-AS3 and miR-340-5p. Results The expression level of HOXA-AS3 in lung cancer tissue was increased significantly (P<0.05) and that of miR-340-5p was decreased significantly (P<0.05). After treatment with various concentrations of cisplatin, the survival rates of DMS114 and DMS114 / DDP cells were decreased gradually (P<0. 05) and the IC₅₀ value of DMS114 / DDP cells was higher than that of DMS114 cells (P<0. 05). Compared with DMS114 cells, the expression level of HOXA-AS3 in DMS114 / DDP cells was increased significantly (P<0.05). Transfection with si-HOXA-AS3 significantly reduced cell viability (P<0.05) and increased the apoptosis rate (P<0.05). Dual luciferase reporter assays confirmed that HOXA-AS3 targeted and bound to miR-340-5p. Cotransfection of si-HOXA-AS3 and anti-miR-340-5p significantly increased cell viability ( P< 0.05) and reduced the apoptosis rate ( P< 0.05 ). Conclusions Interfering with the expression of HOXA-AS3 inhibits cell proliferation and promotes apoptosis, thereby enhancing the sensitivity of DMS114 / DDP cells to cisplatin. The mechanism of action may be related to upregulation of miR-340-5p expression.

Abstract: Objective To investigate the regulatory mechanism of long-chain non-coding RNA prostate cancer- related transcription 1 (PCAT1) in proliferation, migration, invasion and glutamine decomposition of tongue squamous cell carcinoma (TSCC) cells. Methods Real-time quantitative polymerase chain reaction was used to detect expression of PCAT1 and miR-329-3p in TSCC tissues, adjacent tissues, TSCC cells and normal oral keratinocytes. Liposomal transfection was applied to UM1 cells. Groups were the pcDNA group ( transfected with pcDNA), pcDNA-PCAT1 group (transfected with pcDNA-PCAT1), miR-NC group (transfected with miR-NC), miR-329-3p group (transfected with miR- 329-3p mimics), pcDNA-PCAT1 + miR-NC group (cotransfected with pcDNA-PCAT1 and miR-NC) and pcDNA-PCAT1 + miR-329-3p group ( cotransfected with pcDNA-PCAT1 and miR-329-3p mimics). Cell counting kit, migration assay (Transwell) and enzyme-linked immunosorbent assay were used to assess cell proliferation, migration / invasion, and levels of glutamine decomposition products glutamate and α-ketoglutarate (α-KG). The dual luciferase assay was used to detect binding between PCAT1 and miR-329-3p in cells. Results Compared with adjacent tissue, expression of PCAT1 in cancer tissue was upregulated significantly and expression of miR-329-3p was downregulated significantly. Compared with NHOK cells, PCAT1 expression was upregulated and miR-329-3p expression was downregulated in UM1, CAL-27 and SCC25 cells and of (P< 0. 05). Compared with the pcDNA group, expression of PCAT1 was significantly increased in the pcDNA- PCAT1 group, cell proliferation, invasion and migration were upregulated, and the levels of glutamate and α-KG were increased significantly (P<0. 05). PCAT1 targeted miR-329-3p. Compared with the miR-NC group, expression of miR- 329-3p in TSCC cells of the miR-329-3p group was increased significantly cell proliferation, migration, and invasion were reduced significantly, and the levels of glutamate and α-KG were reduced significantly ( P< 0. 05). Compared with the pcDNA-PCAT1+miR-NC group, expression of PCAT1 in the pcDNA-PCAT1+miR-329-3p group was reduced significantly, cell proliferation, migration, and invasion were reduced significantly, and the levels of glutamate and α-KG were also reduced significantly (P< 0. 05). Conclusions PCAT1 promotes the proliferation, migration, invasion, and glutamine decomposition of TSCC cells and its mechanism is related to targeting miR-329-3p.

Abstract: Objective To investigate the inhibitory effects of procaine on the proliferation, migration and invasion of osteosarcoma cells through the extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinase (MAPK) / focal adhesion kinase (FAK) pathway. Methods Human osteosarcoma Saos-2 cells were cultured in vitro and divided into control (without drugs), procaine ( 2 μmol / L procaine), Y15 ( 10 μmol / L Y15) and procaine + Y15 ( 2 μmol / L procaine + 10 μmol / L Y15) groups. Proliferation of Saos-2 cells was assess by the CCK-8 method. Apoptosis of Saos-2 cells was detected by flow cytometry. The migration and invasion of Saos-2 cells were assessed by the transwell method. Expression of ERK/ MAPK/ FAK pathway-related proteins in Saos-2 cells was detected by Western blot. Results Compared with those of the control group, the Saos-2 cell survival rate, migrated cell number, invasive cell number and protein expression levels of p-ERK/ ERK, p-p38 MAPK/ p38 MAPK and p-FAK/ FAK in procaine, Y15 and procaine + Y15 group were significantly lower (P<0. 05) and the apoptotic rate was significantly higher (P<0. 05). Compared with those in the Y15 group, there was no significant difference in the Saos-2 cell survival rate, apoptotic rate, migrated cell number, invasive cell number, or protein expression levels of p-ERK/ ERK, p-p38 MAPK/ p38 MAPK and p-FAK/ FAK in the procaine + Y15 group ( P> 0.05). Conclusions Procaine inhibits the proliferation, migration and invasion of osteosarcoma Saos-2 cells, which may be mediated by inhibiting activation of the ERK/ MAPK/ FAK pathway.

Abstract:Alzheimer’ s disease ( AD) is one of the most common neurodegenerative diseases. Its main pathological markers include neurofibrillary tangle and senile plaque formation, and its pathogenesis is extremely complex. As the main factor of the insulin signaling pathway, Akt plays an important role in regulating cell growth, energy utilization, mitochondrial functions, autophagy, oxidative stress, synaptic plasticity and cognitive functions. Studies have shown different post-translational modifications of Akt in the brains of AD patients, which suggests that different post- translational modifications of Akt may be closely related to the pathogenesis of AD. In this review, the possible role of Akt in the pathogenesis of AD was discussed, in order to provide new clues for the prevention and treatment of AD.

Abstract:With the changes in living conditions of modern society, patients with mental disorders represented by depression and anxiety are increasing yearly. However, the low cure rate of existing treatment measures needs to be improved. Clarifying the pathological mechanisms of these diseases is of great significance for their prevention and treatment. Such models include induced and genetic models. Genetic models have the advantages of a stable phenotype, clear pathological processes and a long treatment window. This article summarizes the genetic models of common mental disorders in rats and mice, briefly introduces the behavioral phenotypic characteristics and provides references for experimental psychiatric research based on animal behavior.

Abstract:Irritable bowel syndrome ( IBS) is a common functional gastrointestinal disorder. The lack of early diagnostic method and effective pharmacotherapy of IBS is predominantly attributed to the current limited understanding of its pathogenesis. MicroRNA (miRNA) is a class of small noncoding RNA of approximately 22 nt in length, which regulate gene expression by post-transcriptional repression or degradation of mRNAs, most often leading to gene silencing. Studies have revealed various differentially expressed miRNAs in colon tissue and peripheral blood of IBS patients. MiRNA has varying degrees of influence on the intestinal flora of IBS patients, the intestinal mucosal barrier, and low grade intestinal inflammation. This review focuses on miRNAs in IBS to find evidence of miRNA involvement in IBS pathogenesis and verify the diagnostic and therapeutic value of miRNA in IBS.

Abstract:Diabetic angiopathy is one of the most serious complications of diabetes. In a state of high glucose, normal functions of blood vessels are damaged, which leads to a series of vascular complications such as diabetic retinopathy, diabetic nephropathy, lower extremity arteriosclerosis occlusion and aortic aneurysm. However, recent studies have revealed that autophagy as an intracellular protein degradation pathway and stress defense mechanism, plays a significant role in the progression of diabetic angiopathy. Thus, we have summarized the progress on research with respect to autophagy-related genes, autophagy signaling pathways and the roles of autophagy in diabetic angiopathy and discuss the detailed mechanisms, which may provide treatment options for diabetic angiopathy.

Abstract:The glomerular filtration rate (GFR) is important in the evaluation of renal clinical indicators, and it is the basis for the definition and staging of chronic kidney disease. In clinical practice, GFR assessment is mainly based on empirical conversion of the serum creatinine value and urea nitrogen, but the sensitivity is low, which is insufficient to diagnose subclinical and critical stage renal failure, cannot evaluate single renal functions. Therefore, there is an urgent need for a non-invasive examination method that quickly and accurately assesses GFR and provides detailed kidney anatomy to avoid a missed diagnosis of early kidney injury. Recent studies have shown that quantitative single-photon emission computed tomography / computed tomography combined with a deep learning automatic segmentation technique, magnetic resonance urography, one-stop computational tomography urography has a great potential for rapid, and comprehensive evaluation of GFR, which may become the main imaging method for clinical GFR evaluation in the future. This article reviews the traditional GFR evaluation method and summarizes the improvement of GFR evaluation in recent years as well as the advantages and disadvantages of new technology analysis.