Abstract: Objective To investigate the effect of stimulants on the expression of T cell activation markers (CD69, CD38, HLA-DR) and a nuclear proliferation marker ( Ki67), and provide the basis for the study of T cell function after activation. Methods Peripheral blood was collected from healthy rhesus monkeys, then peripheral blood mononuclear cells ( PBMCs ) were separated and regular doses of PMA + Ionomycin, α-CD2 / α-CD3 / α-CD28, and phytohemagglutinin-P (PHA-P)+interleukin-2 ( IL-2) were added. Expression of CD4+ T cells and CD8⁺T cell surface markers, CD69, CD38, HLA-DR, and Ki67, were detected at different time points. Results Flow cytometry showed that when the stimulant, α-CD2 / α-CD3 / α-CD28, acted for 72 hours, expression of CD69 and Ki67 on CD4⁺T cells and CD8⁺T cells was higher compared with the other two stimulants, while the percentage of HLA-DR+ CD4⁺T cells and HLA-DR+ CD8⁺T cells and CD38⁺CD8⁺T cells was not different. Meanwhile, the percentage of CD38⁺CD4⁺T cells was not different between α-CD2 / α-CD3 / α-CD28 and PHA-P + IL-2 groups ( P> 0. 05). Expression of all four molecules was very low under PMA+Ionomycin stimulation. Conclusions CD2 / α-CD3 / α-CD28 had the best effect in activating T cells, followed by PHA-P+IL-2, and then PMA+Ionomycin. This provides the experimental basis for selecting appropriate stimulants on the basis of different experimental purposes.

Abstract: Objective Neuroinflammation is a phenotype of leptin receptor (LEPR) mutated mice (db / db) and raises the question of whether LEPR is involved in microglial activation. To examine the function of LEPR on microglia cells, microglial activation was comparatively analyzed in LEPR^{+ /+} and LEPR^-/- rats. Methods RT-PCR, Western blot, immunohistochemistry, and immunofluorescence were used to examine microglia morphology, inflammatory factor secretion, and sensitivity to lipopolysaccharide ( LPS) treatment in vitro and in vivo. Results First, LEPR was expressed in microglia from LEPR^{+ /+} rats, while LEPR protein was completely deleted in microglia from LEPR^-/- rats. LEPR deletion enhanced sensitivity to LPS treatment and reduced survival rate of LEPR^-/- rats by 75%. Activated microglia were significantly increased in brain tissue of LEPR^-/- rats compared with LEPR^{+ /+} rats. LEPR deletion also increased the expression of inflammatory cytokines including interleukin ( IL) -6, inducible nitric oxide synthase, and Interleu bin-1β (IL-1β), and enhanced phagocytotic ability. Phosphatidylinositol-3-kinase (PI3K) / AKT phosphorylation was significantly increased in microglia from LEPR^-/- rats compared with LEPR^{+ /+} rats, suggesting that activation of the PI3K/ AKT signal pathway could partly be the mechanism of microglia activation. Conclusions Deletion of LEPR in microglia induced its activation and suggests the involvement of neuroinflammation in rats. Our result also suggest that the LEPR/ leptin axis plays important roles in neuroinflammation through microglia.

Abstract: Objective We treated simian immunodeficiency virus ( SIV) mac239-infected rhesus macaque monkeys with antiretroviral therapy and examined the characteristics of CD32 expression on resting, activated, and / or memory CD4+ T-cell subsets, to determine their potential as a biomarker of HIV/ SIV reservoirs. Methods Four monkeys chronically infected with SIVmac239 were intramuscularly administrated antiretroviral therapy. Using this animal model, we detected plasma viral loads, CD4⁺T-cell counts, CD4 / CD8 ratios, and the percentage of CD32 expression in different CD4⁺T-cell subsets at different stages. Results Compared with the pre-infection stage, the percentage of CD32 expression in activated naive CD4⁺T cells and HLA-DR⁺ CD4⁺T-cell subsets was significantly increased at the post-infection stage. Moreover, there were no changes in the percentages of CD32 expression in resting CD4⁺T cells, resting naive CD4⁺T cells, resting central memory CD4⁺T cells, resting effector memory CD4⁺T cells, and resting TEMRA CD4⁺T cells. Conclusions This study provides more support for the view that CD32 is not the main biomarker of SIV reservoirs, and provides insight for future AIDS treatment.

Abstract: Objective To investigate the anti-effects of N-acetyl-seranyl-aspartate-lysyl-proline (Ac-SDKP) on experimental silicosis by regulating YEATS domain-containing protein 4 (YEATS4). Methods RAW264. 7 cells were divided into a siRNA-negative control (NC) group (siC), siRNA-YEATS4 group (siY), silicon dioxide (SiO₂ ) + siRNA- NC group (S+siC), and SiO₂ + siRNA-YEATS4 group ( S +siY). In addition, there was a control group ( C), SiO₂ - induced group (S), SiO₂+ Ac-SDKP treatment group ( S+Ac), and AC-SDKP treatment group (Ac). Wistar rats were randomly divided into a control 24-week group (C24), silicosis 24-week group ( S24), and Ac-SDKP treatment 24-week group (Ac24). Expression of collagen type I (COL I), monocyte chemoattractant protein-1 (MCP-1), arginase 1, and YEATS4 were measured by western blotting in lung tissue and RAW264. 7 cells. Immunohistochemical staining was used to detect YEATS4 expression and localization in lung tissue and RAW264. 7 cells. Results Western blot showed down- regulated protein expression levels of COL I, MCP-1, and arginase 1 in the siY and S+siY groups compared with the siC and S+siC groups (P< 0. 05). Compared with the C group, the levels of COL I, MCP-1, arginase, and YEATS4 were increased in the S group. In addition, the levels of COL I, MCP-1, arginase 1, and YEATS4 were reduced in the S+Ac group compared with the Sgroup in RAW264. 7 cells ( P< 0. 05). Immunohistochemistry and Western blot showed that expression of COL I, MCP-1, arginase-1, and YEATS4 was up-regulated in the S24 group compared with the C24 group. Expression of COL I, MCP-1, arginase-1, and YEATS4 in the Ac24 group was decreased compared with S24 silicosis rats (P< 0. 05). Conclusions Ac-SDKP may play a suppressing role in silicosis by inhibiting YEATS4.

Abstract: Objective Cardiac fibroblasts account for more than 50% of heart cells and play an important role in the physiological and pathological processes of the heart. Seven days of age is an important turning point in mice when the heart loses its ability to regenerate. This study analyzed the changes in the number and characteristics of cardiac fibroblasts in the early postnatal period of mice, and provides basic experimental data for the study of the myocardial regeneration mechanism. Methods Apical resection (AR) was performed on 1-day-old and 7-day-old C57 mice. The apex incision was photographed with a stereoscope and stained with hematoxylin and eosin (HE), and regeneration was observed at 21 days post resection. Heart tissues of C57 mice at postnatal day 1 (P1) and P7 were isolated and weighed to compare the wet weight. Cardiac pathological structures at the two time points were observed by HE staining in pathological sections, and fibroblasts were observed by immunofluorescence staining. To accurately quantify the number of cardiac fibroblasts, we used fibroblast surface marker thymocyte differentiation antigen 1 (Thy1) to sort cardiac tissue cell suspensions by flow sorting technology. To investigate changes in cardiac fibroblast characteristics, we isolated mouse cardiac fibroblasts on P1 and P7 and analyzed the mRNA expression of fibroblast marker proteins Thy1, fibroblast-specific protein 1 (Fsp-1), Periostin, platelet-derived growth factor receptor α ( Pdgfrα), collagen alpha-1 ( Col1a1), and transcription factor 21 ( Tcf21). Results Heart tissue of 1-day-old mice could regenerate (regeneration rate was 86. 67%) 21 days after apical resection, whereas that of 7-day-old mice could not regenerate ( regeneration rate was 0). The average wet weight of heart tissue in mice at P7 was 13. 13 mg (3. 11 times) heavier than that at P1 (P1: 6. 22 ± 0. 19, P7: 19. 35 ± 0. 56, P< 0. 0001; n= 6). Immunofluorescence staining indicated that the number of cardiac fibroblasts at P7 was significantly higher than that at P1. Flow cytometry showed that the proportion of cardiac fibroblasts to total cells in heart tissue at P7 was 2. 9% (1. 38 times) higher than that at P1 ( P1: 7. 7 ± 0. 74, P7: 10. 6 ± 0. 95, P= 0. 029; n= 3). At the level of fibroblast transcription, Thy1, Fsp-1, and Periostin expression was higher at P7 than at P1 (Thy1: P1: 1. 01 ± 0. 12, P7: 2. 71 ± 0. 27, P= 0. 0288; Fsp-1: P1: 1. 04 ± 0. 27, P7: 5. 28 ± 0. 10, P= 0. 0046; Periodin: P1: 0. 91 ± 0. 01, P7: 1. 13 ± 0. 05, P= 0. 0119; n= 3), and Pdgfrα, Col1a1, and Tcf21 expression was lower at P7 than at P1 (Pdgfrα: P1: 1. 09 ± 0. 04, P7: 0. 62 ± 0. 01, P= 0. 0068; Col1a1: P1: 1. 00 ± 0. 09, P7: 0. 57 ± 0. 02, P= 0. 0433; Tcf21: P1: 1. 00 ± 0. 03, P7: 0. 54 ± 0. 02, P= 0. 0054; n= 3), which indicated that cardiac fibroblast traits changed during the period from 1 to 7 days after birth. Conclusions The proportion and characteristics of cardiac fibroblasts in mice changed significantly during the early postnatal period (1 to 7 days), which provides some clues to the mechanism of the loss of mammalian myocardial regeneration.

Abstract: Objective To investigate the effects of Naoshuantong capsule on the expression of inflammatory cytokines IL-1β, IL-6, ICAM-1, MMP-9 and TNF-α in the atherosclerosis ApoE^-/-mouse model and to explore therapeutic mechanisms. Methods Eight-week-old ApoE^-/- male mice were fed a high-fat diet to establish atherosclerosis. After the diet, the mice were randomly divided into the atherosclerosis control group, Naoshuantong capsule low-dose group and Naoshuantong capsule high-dose group. The low-dose and high-dose groups were given 0. 5 and 1. 0 g / kg Naoshuantong capsule, respectively, and the control group was given an equivalent volume of distilled water. After 12 weeks, whole-aortic intimal plaques were detected by oil red O staining, and protein levels of inflammation-related factors IL-1β, IL-6, ICAM- 1, MMP-9 and TNF-α were observed and compared between each group. Results After drug intervention, the aortic plaque area of the high-dose group was significantly lower than that of the control group (P< 0. 01). Compared with the atherosclerosis control group, protein expression levels of IL-1β and IL-6 were decreased in the low-dose group ( P< 0. 05), and IL-1β, IL-6, ICAM-1 and TNF-α levels were markedly reduced in the high-dose group (P<0. 05,P<0. 01). The expression of MMP-9 was not significantly different compared with the control group ( P> 0.05). Conclusions Naoshuantong capsule regulated the expression of inflammatory-related factors, and its potential mechanism for the treatment of atherosclerosis may be related to inflammatory-related pathways.

Abstract: Objective To investigate the neuroprotective effect of total flavones of Dracocephalummoldavica L. (TFDM) on MPTP-induced Parkinson’s disease (PD) model mice. Methods After the MPTP-induced PD model was established, the effect of TFDM on motor function was evaluated by Rota Rod and climbing pole performance. Expression of oxidative stress factor superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were detected by ELISA. The expression of tyrosine hydroxylase (TH) and apoptosis-related proteins caspase-3, Bcl-2, and Bax were detected by immunohistochemistry and western blot. Results Behavioral evaluation showed that TFDM therapy significantly increased the fall latency time (P< 0. 05) and decreased the climbing time (P< 0. 05). ELISA showed that TFDM therapy significantly reduced MDA content (P< 0. 01), and the expression of SOD and GSH-Px were significantly increased (P<0.05). Immunohistochemistry and Western blot showed increased TH, Bcl-2, Bcl-2/ Bax protein expression (P< 0. 05), and decreased caspase-3 and Bax expression (P< 0.05). Conclusions TFDM improved motor dysfunction and reduced oxidative stress injury and dopaminergic neuron loss in PD model mice. The neuroprotective effect may be mediated by inhibiting caspase-3 activity, upregulating Bcl-2, and downregulating the expression of Bax.

Abstract: Objective To investigate the role of microRNA (miR)-20a-5p regulating vascular endothelial growth factor (VEGF) pathway in mice with oxygen-induced retinopathy (OIR). Methods The experiment was divided into normal group, model group, hyperoxia control group, miR-20a-5p high expression group, with 24 mice in each group. In addition to the normal group, the rest of the mice were placed in the oxygen tank with ( 75. 00 ± 2. 00)% oxygen concentration from the 7th day after birth to establish the OIR mice model, and after 5 days of continuous hyperoxia environment, they were fed in normoxia, at 1 day before the end of hyperoxia environment, 1 μL phosphate buffered saline (PBS) was injected into the vitreous cavity of the hyperoxia control group, 1 μL miR-20a-5p agomir (1 μmol / L) was injected into the vitreous cavity of the high expression group, and the model group did nothing. After the end of hyperoxia, the normal group was kept in the air for normal feeding. The experiment was carried out after normal air feeding for more 5 days. The expressions of miR-20a-5p, VEGF, vascular endothelial growth factor receptor (VEGFR)-1 and VEGFR-2 were detected by real-time fluorescence quantitative PCR ( qRT-PCR); retinal vascular morphology was observed by retinal patch; hematoxylin eosin (HE) staining was used to count the endothelial cells of retinal neovascularization; and VEGF positive cells were detected by immunohistochemistry. Results In the model group and hyperoxia control group, on the 17th day of life, the radial large blood vessels from the optic disc extended roundly and irregularly, there were a lot of new blood vessels, the structure and distribution of neovascularization were disordered and the capillary network was occluded; and compared with the model group and the hyperoxia control group, the miR-20a-5p high expression group had no obvious radial vascular circuitous, less irregular vascular expansion and less neovascularization. Compared with those in the normal group, the level of miR-20a-5p in retina in model group and hyperoxia control group was lower (P< 0. 05), and the number of nuclei in retinal vascular endothelium, VEGF protein positive area percentage, expressions of VEGF, VEGFR-1 and VEGFR-2 mRNAs in retina were higher (P< 0. 05); meanwhile, compared with those in the model group and the hyperoxia control group, the level of miR-20a-5p in retina in miR-20a-5p high expression group was lower (P< 0. 05), and the number of nuclei in retinal vascular endothelium, VEGF protein positive area percentage, expressions of VEGF, VEGFR-1 and VEGFR-2 mRNAs in retina were higher ( P< 0. 05). Conclusions Increasing miR-20a-5p can inhibit VEGF pathway and decrease retinal neovascularization in OIR mice, so as to protect the retina of OIR mice.

Abstract: Objective To examine the effect of sevoflurane analgesia during hypoxia in neonatal rats on the mitogen-activated protein kinase ( MAPK) signaling pathway. Methods In the model group and sevoflurane group, pregnant rats were administered birth asphyxia models. The model group was administered 50% oxygen by volume, while the sevoflurane group was administered 50% oxygen and 2. 5% sevoflurane. The natural delivery group gave birth spontaneously. The learning and memory abilities of newborn ofFspring were assessed. Serum superoxide dismutase (SOD), malondialdehyde (MDA) levels, and number of Nissl bodies in the CA1 area of the hippocampus in brain tissue were examined. Brain tissue MAPK, B-cell lymphoma-2 gene (Bcl-2), Bcl-2 related X protein (Bax), and cysteine protease-3 (caspase-3) mRNA and protein expression were also examined. Results Compared with the model group, newborn ofFspring in the sevoflurane group showed a longer residence time in the target quadrant, increased number of crossing platforms, increased serum SOD, decreased MDA, increased number of Nissl bodies in the hippocampal CA1 area, and increased expression of Bcl-2 mRNA and protein in brain tissue. Expression of MAPK, Bax, and caspase-3 mRNA and protein was decreased (P< 0.05). Conclusions Sevoflurane labor analgesia can effectively improve hypoxic-ischemic brain damage in neonatal rats. It may play a protective role in the brain by inhibiting the MAPK signaling pathway and in turn inhibiting hippocampal neuron damage.

Abstract: Objective To examine transcription levels of vitamin D metabolic enzymes and the vitamin D receptor (VDR) in the testes of adult tree shrews. Specifically, to detect protein expression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) and investigate causes of reproductive disorders in male tree shrews. Methods Male tree shrews (aged 2 to 3 years) were selected. On the basis of individual records, shrews were divided into a normal group with a successful reproductive history and a reproductive disorder group without a reproductive history; there were five shrews in each group. One side of the testis was surgically removed and histopathological changes were observed by pathology. Real- time fluorescent quantitative PCR was used to detect changes in mRNA levels of VDR and vitamin D metabolizing enzymes in the testis. Protein expression of CYP24A1 was detected by western blotting. Expression of sperm CYP24A1 was detected by immunofluorescence. Results In the normal group, the seminiferous tubules were tightly arranged and there were more spermatogenic cells than in the reproductive disorder group. Compared with the normal group, there was no significant difference in mRNA levels of VDR, CYP2R1, or CYP27A1 in the testes of tree shrews (P> 0. 05). However, mRNA expression levels and protein levels of CYP24A1 were significantly lower than in the normal group (P< 0. 05). At the same time, immunofluorescence showed that sperm from the testis of the normal group had high expression of CYP24A1 in the head, neck, and midpiece, while the reproductive disorder group had low or no expression of CYP24A1. Conclusions CYP24A1 is directly related to successful fecundity of male tree shrews, suggesting that CYP24A1 can be used as an important index to evaluate the fecundity of male tree shrews.

Abstract: Objective To investigate the effect of α-mangostin on immune function in a cyclophosphamide (CTX)-induced immunosuppressive mouse model, so as to provide experimental basis for further study of the mechanism of α-mangostin and its potential for development as a new drug to enhance immunity. Methods Mice were randomly divided into five groups: normal control group, immunosuppressive group, and high, middle, and low dose groups. All groups except the normal control group were given CTX to create the immunosuppressive model in the first 7 days. In the latter 14 days, the normal control group was given normal saline, the immunosuppressive group was given corn oil, and the three dose groups were given different doses of α-mangostin. The mice were killed 24 h after the last treatment. We assessed the effect of α-mangostin on immune function in mice by detecting delayed allergic reaction ( DTH), serum hemolysin ( HC50), macrophage phagocytosis (carbon clearance test), thymus and spleen index of immune organs, natural killer cell (NK cell) activity (MTT method ), peripheral white blood cell count, and splenic lymphocyte proliferation. Results The immunosuppressive mouse model was established by intragastric administration of CTX. After administration, the thymus and spleen index, hemolytic value, spleen lymphocyte proliferation rate and NK cell activity in immunosuppressive mice were increased by different doses of α-mangostin. The effect was most prominent in the high dose group, in which the total dose of α-mangostin was [100 mg (kg,d)^-1 ]. In many experiments, the difference was statistically significant compared with the immunosuppressive group (P< 0. 01). In the delayed allergic reaction, there was significant difference between the high dose group and the normal control group and immunosuppressive group (P< 0. 05), but there was no significant difference between the other groups (P> 0. 05). There was a significant difference in peripheral white blood cell counts between the high dose group and the immunosuppressive group (P< 0. 01), but no conspicuous difference between the other groups ( P> 0. 05 ). Macrophage phagocytosis, measured by carbon clearance assay, was similar in the immunosuppressive group and each dose group ( P> 0. 05). Conclusions mangostin had a dose-dependent regulating effect in immunosuppressed mice, and the high dose of α-mangostin demonstrated the strongest improvement of immune function in immunosuppressed mice.

Abstract: Objective To explore the promotive effect and mechanism of human cord blood mononuclear cells (HCMNCs) on angiogenesis in rats with acute myocardial infarction ( AMI). Methods HCMNCs were isolated and labeled with BrdU. Twenty rats were selected to establish the acute myocardial infarction model by coronary artery ligation. The rats were randomly divided into myocardial infarction and HCMNC groups with 10 rats in each group. Another 10 rats were used as the sham operation group. After successful model establishment, HCMNCs were injected around the myocardial infarction in the HCMNC group and L-DMEM was injected into the same site of sham operation and model groups. Four weeks after transplantation, echocardiography was used to assess cardiac functions, HE staining was used to observe myocardial pathological changes, BrdU staining was used to detect transplanted cell survival, and immunohistochemical staining was used to analyze the microvessel density (MVD) and protein expression of CD31 vascular endothelial growth factor (VEGF). Western blott was used to measure CD31 and VEGF protein levels in myocardial tissue. Results Compared with the sham operation group, the left ventricular end systolic dimension ( LVEDs ) and left ventricular end diastolic dimension (LVEDd) were increased and the left ventricular ejection fraction (LVEF) and fraction shortening (FS) were decreased in myocardial infarction and HCMNC groups (P<0. 05). Compared with the myocardial infarction group, LVEDs and LVEDd were decreased, and LVEF and FS were increased in the HCMNC group (P<0. 05). HE staining showed that the structure of myocardial cells was normal in the sham operation group and the arrangement of myocardial cells was disordered in the myocardial infarction group with a large amount of inflammatory cell infiltration. The structure of myocardial cells in the HCMNC group was essentially normal. BrdU-positive cells were not detected in sham operation or myocardial infarction groups and scattered BrdU-positive cells were seen in the infarct area of the HCMNCs group, which were involved in formation of the blood vessel wall. Compared with the sham operation group, relative expression of CD31 and VEGF proteins and MVD were decreased in myocardial infarction and HCMNC groups (P<0. 05), and compared with the myocardial infarction group, relative expression of CD31 and VEGF proteins and MVD were increased (P< 0. 05). Conclusions HCMNCs promote the establishment of collateral circulation in AMI rats, induce angiogenesis, and significantly improve ischemic heart functions.

Abstract: Objective We used plaster immobilization to establish different stages of the knee osteoarthritis model to provide an appropriate animal model for precise treatment of acupuncture and traditional Chinese medicine. Methods A total of 40 large-eared white rabbits were selected and randomly divided into model groups ( 1, 2, 3, and 4), with a sample size of 10 in each group. The left hind limbs were immobilized in the extended position. Immobilization lasted for 10 days, 20 days, 30 days, and 40 days, respectively. The behavior, morphology, and pathological manifestations of the rabbits’ knee joints were observed. Results Lequesne myasthenia gravis behavior scores and Jessica Badendick general morphology scores changed significantly in each model group (P< 0. 05). The pathological test result showed a significant phase and difference among the groups. Conclusions In the process of modeling knee osteoarthritis, there are significant differences between the groups. According to the most significant manifestations of each stage, the disease can be roughly divided into an early stage, middle stage, and late stage. Intervention of acupuncture or traditional Chinese medicine may be effective in treating the disease.

Abstract: Objective To investigate the role of microRNA ( miR) -20a-5p in the regulation of the vascular endothelial growth factor (VEGF) pathway in mice with oxygen-induced retinopathy ( OIR) . Methods Mice were divided into a normal group, model group, hyperoxia control group, and miR-20a-5p high expression group, with 24 mice in each group. With the exception of the normal group, the mice were placed in an oxygen tank with ( 75. 00 ± 2. 00) % oxygen concentration beginning postnatal day 7 to establish the OIR model, and after 5 days of continuous hyperoxia environment, they were returned to normal air conditions. At 1 day before the end of the hyperoxia environment, 1 μL phosphate buffered saline was injected into the vitreous cavity of the hyperoxia control group, 1 μL miR-20a-5p agomir ( 1 μmol / L) was injected into the vitreous cavity of the high expression group, and the model group received nothing. The normal group was kept in normal air conditions. The experiment was carried out after the mice were exposed to normal air for 5 more days. The expressions of miR-20a-5p, VEGF, VEGF receptor (VEGFR) - 1 and VEGFR-2 were detected by real-time fluorescence quantitative PCR, retinal vascular morphology was observed by retinal patch, hematoxylin and eosin staining was used to count the endothelial cells of retinal neovascularization, and VEGF-positive cells were detected by immunohistochemistry. Results In the model group and hyperoxia control group, on postnatal day 17, the large radial blood vessels extending from the optic disc were round and irregular. There were many new blood vessels; the structure and distribution of neovascularization were disordered and the capillary network was occluded. Compared with the model group and the hyperoxia control group, the miR-20a-5p high expression group had no obvious circuitous radial vasculature and had less irregular vascular expansion and neovascularization. Compared with those in the normal group, the retinal miR-20a-5p levels in the model group and hyperoxia control group were lower ( P< 0. 05) , and the number of nuclei in retinal vascular endothelium, VEGF protein-positive area percentage, and expressions of VEGF, VEGFR-1 and VEGFR-2 mRNAs in retina were higher ( P< 0. 05) . Compared with those in the model group and the hyperoxia control group, the retinal miR-20a-5p level in the miR-20a-5p high expression group was lower ( P< 0. 05 ) , and the number of nuclei in retinal vascular endothelium, VEGF protein-positive area percentage, and expressions of VEGF, VEGFR-1 and VEGFR-2 mRNAs in retina were higher ( P< 0. 05) . Conclusions Increasing miR-20a-5p inhibited the VEGF pathway and decreased retinal neovascularization in OIR mice, which may protect the retina of OIR mice.

Abstract: Objective To explore the anti-tumor activity of lobaplatin in colon cancer cells, to elucidate the underlying molecular mechanisms, and to provide theoretical evidence for clinical application. Methods Colon cancer cell line SW480 was cultured in vitro. MTT colorimetric assay and colony formation assay were used to detect the inhibitory effect of different concentrations of lobaplatin on colon cancer SW480 cells at 24, 48 and 72 h. The apoptosis rate of the cells was detected by flow cytometry. Western blot was used to detect the expression of the apoptosis-related proteins caspase-3, BAD and BCL-2 in SW480 cells after lobaplatin treatment. The mechanism of lobaplatin-induced apoptosis was investigated by adding AKT inhibitors and agonists and detecting the expression levels of caspase-3, AKT, pAKT, BAD, p-BAD and BCL-2. Results Lobaplatin significantly inhibited the growth of cultured SW480 cells in a dose- and time- dependent manner. Lobaplatin induced apoptosis of colon cancer SW480 cells. Lobaplatin induced downregulation of anti- apoptotic BCL-2 protein expression and upregulated pro-apoptotic BAD and caspase-3 protein expression in the cells. Addition of AKT inhibitor MK - 2206 significantly decreased pAKT, p-BAD, and BCL-2 expression, and significantly increased caspase-3 expression. Addition of AKT agonist SC97 significantly increased pAKT, p-BAD, and BCL-2 protein expression, and significantly decreased caspase-3 protein expression. Conclusions Lobaplatin significantly inhibited proliferation and induced apoptosis of human colon cancer SW480 cells. Lobaplatin may play a role in promoting apoptosis through the AKT/ BCL-2/ BAD signaling pathway, and it is an effective anti-colon cancer drug.

Abstract: Objective The aim of this study was to examine the effect of short-term low-frequency electrical stimulation (SLES) on nerve regeneration of 8-week delayed peripheral nerve injury with a long gap. Methods Left sciatic nerves of adult female Sprague Dawley rats were transected. Next, 8-week-delayed defective nerve models were prepared followed by trimming of the nerve stumps. SLES was applied to the experimental group after the nerve defects had been bridged by contralateral normal sciatic nerves. The nerve defects were also bridged without SLES in the control group. After the animals had been fed for different times as required, tissue specimens were harvested to examine expression of endogenous neurotrophic factors and morphological behavior of regenerating nerves through histological techniques such as Meyer’s trichrome staining, immunofluorescent double-staining, retrograde tracing, and electron microscopy. Results Expression levels of endogenous neurotrophins were higher and more rapidly observed following electrical stimulation. No improved performance of nerve regeneration was found in either development of nerve fibers or reinnervation of target organs. Conclusions The ability of SLES to promote nerve regeneration after a long delay (more than 8 weeks) was limited.

Abstract: Objective To examine the protective effect of pancreatic kininogenase on retinopathy in diabetes mellitus rats, and explore its mechanism of action. Methods Sixty specific-pathogen-free rats were randomly divided into a blank group, model group, and treatment group, with 20 rats in each group. The model group and treatment group were given a single intraperitoneal injection of 60 mg / kg streptozotocin to establish a rat diabetes model. Rats in the blank group were intraperitoneally injected with an equivalent amount of 0. 1 moL/ L sodium citrate. Rats in the treatment group were treated by intraperitoneal injection of 0. 8 U of pancreatic kininogenase, while the blank group and model group were intraperitoneally injected with 0. 2 mL of normal saline once a day for 2 weeks. Hematoxylin and eosin staining was used to observe pathological changes of retinal ganglion cells ( RGCs). Apoptosis of RGCs was detected by TUNEL staining. Activity of superoxide dismutase ( SOD) and malondialdehyde ( MDA) content in the retina were detected by enzyme- linked immunosorbent assay. mRNA and protein expression of Notch1 and HES-1 were detected by quantitative RT-PCR and western blotting. Results In the blank group, the structure of each layer of retinal tissue was clear, RGC nuclei were clear and orderly, and apoptotic cells were only occasionally seen. Compared with the blank group, RGCs in the model group were arranged disorderly, the nuclei were sparse, the number of RGCs was significantly reduced, apoptosis rate was increased, SOD activity of retinal tissue was decreased, and MDA content was increased (P< 0. 05). Compared with the model group, the structure of each layer of retinal tissue in the treatment group was clear, the nuclei of RGCs were clear and regular, the number of RGC layer cells was increased, apoptosis rate was decreased, SOD activity of retinal tissue was increased, and content of MDA was decreased (P< 0. 05). The quantitative RT-PCR and western blotting result showed that mRNA and protein expression levels of Notch1 and Hes1 were decreased in the model group compared with the blank group (P< 0. 05). Compared with the model group, mRNA and protein expression levels of Notch1 and Hes1 in the treatment group were increased ( P< 0. 05). Conclusions Pancreatic kininogenase can protect and improve diabetic retinopathy. The mechanism may be related to the activation of the Notch1 / HES-1 signaling pathway.

Abstract: Objective To generate myristoylated alanine-rich C-kinase substrate-like 1c ( Marcksl1 ) gene knockout mice and investigate the gene function in hematopoiesis. Methods We used CRISPR/ Cas9 technology to produce Marcksl1 gene knockout mice, which was confirmed by PCR and Sanger sequencing. Germline transmission of Marcksl1 gene knockout mice was confirmed by crossing to wild-type mice. We also analyzed the ratio of homozygous, heterozygous, and wild-type mice at different stages of embryonic development. We isolated mice fetal liver to examine the effect of Marcksl1 deletion on the hematopoietic system using routine blood and flow cytometry method . Results PCR and sequencing data showed that Marcksl1 knockout mice were successfully obtained. Our data further suggest that embryonic lethality caused by Marcksl1 gene knockout occurs at a late period of embryonic development. Routine blood and flow cytometry result showed that at E15. 5, deletion of the Marcksl1 gene did not affect the number of white blood cells, red blood cells, or platelets, but the proportion of hematopoietic stem cells in fetal liver increased significantly. Conclusions We have successfully constructed a Marcksl1 knockout mouse. We further found that Marcksl1 deletion increased the number of hematopoietic stem cells. Our work provides a mouse model for further study of the gene function of Marcksl1 in embryonic development and the hematopoietic system.

Abstract: Objective To establish a subcutaneous tumor model in nude mice using patient-derived tissues (PDTs) of lung squamous cell carcinoma (LSCC) and determine the sensitization effect of anlotinib on radiofrequency ablation (RFA)-mediated inhibition of subcutaneous LSCC growth. Methods Patient-derived LSCC tissue was collected and qPCR was used to detect the expression of the target of anlotinib. Then, the tissue was subcutaneously implanted into nude mice to form tumor tissues. Oral administration of anlotinib and RFA treatment of subcutaneous tumors were performed, and qPCR was used to detect epithelial-mesenchymal transition of LSCC. Results Subcutaneous tumor tissue in nude mice was generated from patient-derived LSCC tissues. The target of anlotinib was clearly expressed. RFA effectively killed subcutaneous LSCC tumor tissues. Anlotinib affected RFA by inhibiting EMT in tumor tissue to sensitize LSCC tissues to killing. Conclusions Anlotinib increases the killing effect of RFA on patient-derived LSCC tissues in a nude mouse model, which may facilitate the diagnosis and treatment of LSCC.

Abstract: Objective To investigate the effect of xinan nasoyuan fangliu extract on the apoptosis of olfactory neurons in rats with acute sinusitis and the role of the SDF-1/ CXCR4 signaling pathway. Methods Forty Sprague Dawley rats were divided into a control group, model group, treatment group and inhibitor group, with 10 rats in each group. The rats in all groups except the control group were treated with staphylococcus aureus suspension to establish the acute sinusitis model, and the control group received sterile saline. On the seventh day after the model was established, treatment administration was conducted by gavage every day for 1 week continuously. The treatment group was given xin ′an nasoyuan fangliu extract according to body weight ( 7 g / kg), the inhibitor group received 1 mg / kg CXCR4 specific inhibitor AMD3100, and the control group and the model group were given the same amount of normal saline. Pathological features of sinusitis were observed and scored to confirm the establishment of the model. Enzyme-linked immunoassay was used to detect the expression of inflammatory cytokines TNF-α and IL-1β in the olfactory epithelium tissue. TUNEL staining, to detect the number of apoptotic cells, and immunohistochemistry of SDF-1 and CXCR4 were performed in olfactory epithelial tissue of rats in each group. Western blot was used to assess the expression of pro-apoptotic proteins, such as cleaved caspase 3 and Bax, and anti-apoptotic protein Bcl-2 in neurons of the olfactory epithelium. Results The pathological score of sinusitis in the model group was significantly higher than that in the control group (P< 0. 05), and that in the treatment group was significantly lower than that in the model group (P< 0.05). The expression of inflammatory factors in the model group was significantly increased compared with that in the control group (P< 0. 05), and that in the treatment group was significantly lower than that in the model group ( P<0.05). TUNEL staining showed that olfactory epithelial neuron apoptosis increased in the model group compared with the control group (P< 0.05), and was significantly lower in the treatment group and inhibitor group than in the model group (P< 0.05). In the model group, Bcl-2, SDF-1 and CXCR4 expressions were significantly decreased (P< 0.05), whereas cleaved caspase 3 and Bax expressions were significantly increased (P< 0.05) compared with the control group. Bcl-2, SDF-1 and CXCR4 expressions in the treatment group were significantly increased compared with those in the model group (P< 0.05), whereas cleaved caspase 3 and Bax expressions were significantly decreased in the treatment group (P< 0.05). Conclusions Xinan nasoyuan fangliu extract improved apoptosis of rat superior olfactory neurons in acute sinusitis, and the effect may be mediated by inhibition of the SDF-1/ CXCR4 signaling pathway.

Abstract: Objective To provide a straightforward fixation device and method for needle warming moxibustion treatment on rats. Methods A coat was made for the rat from old cloth. Using the natural tendency of rats to like darkness and seek holes, the rat was bound in the coat. Bound rats were then hung on a cloth strip with S-shaped hooks to provide a swing, which completely exposed the acupoints for treatment. Rats were treated with needle warming moxibustion. We compared operation time and animal reaction between swinging rats bound with coat and bound rats only. Results Compared with the bondage method , the swinging method with rats bound with coat fixation required only one person to complete the operation, and the time required to fix one rat was significantly shorter (P< 0. 01). There was a minimal stress reaction in the process of binding, hanging, and needle warming moxibustion treatment. The needle drop rate was 10%, while in the bound group it was 60%. There was no scalding or death in the swinging rats. Thus, the needle warming moxibustion treatment was successfully administered. Conclusions Compared with the existing holder, the fixation method of needle warming moxibustion treatment on swinging rats bound with coat can reduce the stress response of rats and minimize manpower. It is easy to adjust the size of device, convenient to select acupoints, and is easy to operate. It is a more applicable method for needle warming moxibustion in the limbs of all types of rats.

Abstract:Studies on diagnoses and treatments based on microRNAs ( miRNA/ miR) are increasing. However, currently most studies in the field of cardiovascular diseases are limited to the application of miRNAs in the left ventricle, and few studies have involved the right ventricle. Therefore, this review examines the latest literature and summarizes the application of miRNAs in right heart disease. We first review intraventricular-specific expression of miRNAs and their application in right heart-related disease. Second, the application status of circulating miRNAs in patients with pulmonary hypertension and right ventricular failure is discussed. Finally, the potential of miRNA modulators in the treatment of pulmonary hypertension and their beneficial effects on the right ventricle are described. We hope that the continued accumulation of evidence on miRNAs in the right heart will help improve treatment for patients with pulmonary hypertension and right ventricular disease.

Abstract:The hollow fiber tumor model, also known as the hollow fiber assay (HFA), was designed by the US National Cancer Institute for the purpose of evaluating preliminary in vivo drug efficacy in a simple, rapid manner. In recent years, it has been widely used in the research and development of anti-tumor drugs, from which new and improved optimizations and applications have emerged. This paper systematically summarizes the characteristics and advantages of the HFA in anti-tumor drug screening, together with the last 10 years of progress in HFA applications. We anticipate that the HFA will play an increasingly important role in anti-tumor drug research, and the improved model of HFA will have broad applications in research on individualized cancer treatments and mechanistic studies.

Abstract:As a widely used class of clinical drugs, glucocorticoids play an essential role in the treatment of various diseases. Autophagy maintains homeostasis of the intracellular environment. In recent years, many studies have shown that glucocorticoids can alter the level of autophagy and treat disease. However, side effects often follow the curative effect. In this article, we review the effect of glucocorticoids on autophagy levels and the mechanisms by which glucocorticoids can treat disease through autophagy. We also provide new ideas for further reducing the side effects of glucocorticoids in the treatment of disease.