RAS-selective lethal small molecule 3 inhibits the fibrosis of pathological scar fibroblasts / 中国组织工程研究

BACKGROUND:Abnormal extracellular matrix accumulation and excessive proliferation of fibroblasts are the main manifestations of pathological scars.Excessive proliferation of fibroblasts leads to the production of large amounts of collagen-based extracellular matrix.Therefore,to investigate the role of fibroblast fibrosis in the formation of pathological scar will provide a new idea for revealing the mechanism of pathological scar and biological therapy. OBJECTIVE:To investigate the effect of RAS-selective lethal small molecule 3(RSL3)on the fibrosis of human pathological scar fibroblasts. METHODS:Then cases of pathological scar tissue and normal skin tissue samples from the same individuals,provided by the Department of Burn Plastic Surgery,General Hospital of Ningxia Medical University,were collected.Fibroblasts of human pathological scar and human normal skin were extracted and used in the following experiments.The general condition of the pathological scar tissue and the normal skin tissue was detected by hematoxylin-eosin staining.The appearance of fibroblasts from pathological scar and normal skin were observed by inverted microscope.The fibroblasts were verified by immunofluorescence assay.The cells were treated with different concentrations of RSL3(1,3,5,7,9,11,13 μmol/L).The inhibitory concentration of RSL3 on fibroblasts was detected by cell counting kit-8.Control group(without treatment)and RSL3 intervention group(treated with 7 μmol/L RSL3 for 24 hours)were set up.The mRNA and protein expressions of glutathione peroxidase 4,type Ⅰ collagen,type Ⅲ collagen and α-smooth muscle actin were detected by Qrt-PCR and western blot,respectively.Level of malondialdehyde in cells was detected.The residual scratch area was measured by cell scratch test after 24 hours to calculate the percentage of residual scratch area. RESULTS AND CONCLUSION:The expression of glutathione peroxidase 4 in the pathological scar group was higher than that in the normal skin group(Mrna:t=3.252,P<0.01;protein:t=5.075,P<0.01).The expression of glutathione peroxidase 4 in the pathological scar fibroblast group was higher than that in the normal skin fibroblast group(Mrna:t=10.32,P<0.01;protein:t=26.22,P<0.01).Compared with the control group,the expression of glutathione peroxidase 4 was decreased(Mrna:t=2.798,P<0.05;protein:t=4.643,P<0.01),the content of malondialdehyde was increased(t=2.917,P<0.05),the expression of type Ⅰ collagen(Mrna:t=15.84,P<0.01;protein:t=4.610,P<0.01),type Ⅲ collagen(Mrna:t=28.86,P<0.01;protein:t=7.713,P<0.01)and α-smooth muscle actin(Mrna:t=2.671,P<0.05;protein:t=7.417,P<0.01)were decreased in the RSL3 intervention group.Compared with the control group,the migration ability was weakened in the RSL3 intervention group(t=14.06,P<0.01).To conclude,RSL3 can inhibit the expression of glutathione peroxidase 4 and then inhibit the ability of fibrosis and migration of pathological scar fibroblasts.

Bone morphogenetic protein-2 mediated homocysteine promotes vascular calcification / 中国组织工程研究

BACKGROUND:There is an internal relationship between hyperhomocysteinemia and vascular calcification.However,the pathogenesis of hyperhomocysteinemia promoting vascular calcification is still unclear. OBJECTIVE:To investigate the role of bone morphogenetic protein-2 in hyperhomocysteinemia-induced vascular calcification. METHODS:Human carotid wax samples were divided into a calcified group(n=29)and a non-calcified group(n=13)according to the presence or absence of calcified plaque.Sixteen ApoE-/-mice were randomly divided into a control group and a hyperhomocysteinemia group,with 8 mice in each group.Bone morphogenetic protein-2 vector was used to transfect rat thoracic artery smooth muscle A7r5 cells,and gradient concentration of homocysteine(50,100,200,and 400 μmol/L)was utilized to treat A7r5 cells.Calcification was detected by alizarin red staining and hematoxylin-eosin staining.The interaction of bone morphogenetic protein 2 with Runt-related transcription factor 2 was detected by immunofluorescence,and the expressions of bone morphogenetic protein 2,Runt-related transcription factor 2,and α-smooth muscle actin were detected by immunohistochemistry and western blot assay. RESULTS AND CONCLUSION:(1)Human carotid artery tissue staining revealed that compared with the non-calcification group,inflammatory cells increased and calcification positive rate increased in the calcification group(P<0.05).Compared with the non-calcification group,the expressions of bone morphogenetic protein-2 and Runt-related transcription factor 2 were up-regulated,and the expression of α-smooth muscle actin was decreased in the calcification group(all P<0.05).(2)The staining of mouse arterial specimens exhibited that,the positive rate of calcified area in the hyperhomocysteinemia group was significantly higher than that in the control group(P<0.05);serum homocysteine level in the hyperhomocysteinemia group was significantly higher than that in the control group(P<0.05).Compared with the control group,the expressions of bone morphogenetic protein-2 and Runt-related transcription factor 2 were up-regulated,and the expression of α-smooth muscle actin was decreased in the hyperhomocysteinemia group(all P<0.05).(3)A7r5 cell culture analysis demonstrated that with the increase of homocysteine concentration gradient,the degree of calcification,the content of bone morphogenetic protein-2 and Runt-related transcription factor 2 protein in A7r5 cells increased(P<0.05),and the content of α-smooth muscle actin protein decreased(P<0.05).(4)The A7r5 cell culture analysis of overexpressed bone morphogenetic protein 2 showed that the calcification degree of the overexpressed bone morphogenetic protein 2 group was increased compared with the corresponding control group,the β-sodium glycerophosphate group,and the homocysteine group.RUNt-related transcription factor 2 expression up-regulated(P<0.05)and α-smooth muscle actin expression down-regulated(P<0.05).(5)The expression of bone morphogenetic protein 2 increased in A7r5 cells cultured with homocysteine in calcified medium,and the expression of Runt-related transcription factor 2 increased with the increase of bone morphogenetic protein 2 expression.(6)The results confirm that bone morphogenetic protein-2 is a key target gene in the regulation of smooth muscle cell phenotypic transformation resulting in vascular calcification by hyperhomocysteinemia.Targeted regulation of bone morphogenetic protein-2 reduces hyperhomocysteinemia-induced vascular calcification.

The expression and diagnostic value of RARRES2, MACF1, and DCN in cerebrospinal fluid of patients with neurosyphilis / 中国医师杂志

Objective:To investigate the expression of retinoic acid receptor responsive gene 2 (RARRES2), microtubule microfilament cross-linking factor 1 (MACF1), and core protein polysaccharide (DCN) in cerebrospinal fluid (CSF) of patients with neurosyphilis, and their diagnostic value for neurosyphilis.Methods:A total of 64 non neurosyphilis syphilis patients (syphilis group) and 78 neurosyphilis patients (neurosyphilis group) admitted to the Second Hospital of Nanjing between June 2020 and September 2022 were included. Among neurosyphilis patients, there were 48 early neurosyphilis patients (early group) and 30 late neurosyphilis patients (late group). Patients with neurosyphilis are treated with routine symptomatic therapy and antibiotic therapy to expel syphilis. The mRNA levels of RARRES2, MACF1, and DCN in CSF of patients with neurosyphilis before and after treatment were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The National Institutes of Health Stroke Scale (NIHSS) was used to evaluate the neurological function of patients with neurosyphilis before and after treatment. The diagnostic value of various indicators for neurosyphilis was analyzed using receiver operating characteristic (ROC) curves.Results:The mRNA levels of RARRES2, MACF1, and DCN in CSF of patients with neurosyphilis were higher than those in the syphilis group (all P<0.001). The mRNA levels of RARRES2, MACF1, and DCN in the CSF of patients with advanced neurosyphilis were higher than those in the early group (all P<0.001). Compared with before treatment, the NIHSS score and RARRES2, MACF1, and DCN mRNA levels of neurosyphilis patients decreased after treatment (all P<0.001). The area under the curve (AUC), sensitivity, and specificity of the combined diagnosis of RARRES2, MACF1, and DCN mRNA in CSF for neurosyphilis were 0.995%, 100.00%, and 93.75%, respectively. The AUC and sensitivity were higher than those of individual diagnosis. Conclusions:The expression of RARRES2, MACF1, and DCN is elevated in CSF of patients with neurosyphilis, and is associated with disease severity and treatment response. These three genes may be candidate biomarkers for diagnosing neurosyphilis.

Study of Improvement Effects and Mechanism of Saposhnikovia Divaricata Extract on IgE Sensitized RBL-2H3 Cells / 浙江中医药大学学报

[Objective]To explore the degranulation effect and mechanism of Saposhnikovia divaricata(SD)extract on rat basophilic leukemia cell line RBL-2H3 cell.[Methods]Methylthialazole tetrazolium(MTT)test was used to select the concentrations in the subsequent experiments based on impact of 5,25,50,100,200,400 μg·mL-1 SD extract on the activity of RBL-2H3 cells.Immunoglobulin E(IgE)induction was used to establish RBL-2H3 cell degranulation model.Blank control group,model group,low dose SD extract group(5 μg·mL-1),medium dose SD extract group(25 μg·mL-1),high dose SD extract group(50 μg·mL-1)and dexamethasone(DXMS)group(100 μg·mL-1)were set up,with intervention for 30 minutes.MTT test was used to detect the effect of low,medium,high-dose SD extract on activity of RBL-2H3 cell degranulation model.Toluidine blue staining was used to observe the morphology of degranulation cells and calculate degranulation rates.Immunofluorescence staining was used to detect expression of F-actin.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of β-aminohexosidase,histamine,interleukin-4(IL-4),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ).Western blot was used to detect the expressions of phosphatidylinositide-3 kinase(PI3K),phosphorylation-PI3K(p-PI3K),protein kinase B(AKT),phosphorylation-AKT(p-AKT),p38 mitogen activated protein kinase(p38MAPK),phosphorylation-p38MAPK(p-p38MAPK),nuclear factors-κB(NF-κB),phosphorylation-NF-κB(p-NF-κB),extracellular regulated kinases(ERK)and phosphorylation-ERK(p-ERK)protein.[Results]The low,medium,high doses of SD extract(5,25,50 μg·mL-1)had no significant effects on the activity of RBL-2H3 cells(P＞0.05).Compared with blank control group,the number of toluidine blue stained cells of model group was decreased,cells shape rounded,degranulation rate was increased,expression of F-actin was decreased,the levels of β-aminohexosidase,histamine,IL-4,IL-6,TNF-α were increased,IFN-γ level was decreased,the expressions of p-PI3K/PI3K,p-AKT/AKT,p-p38MAPK/p38MAPK,p-NF-κB/NF-κB and p-ERK/ERK were increased(P＜0.01).Compared with model group,the F-actin expression of low,medium,high doses of SD extract groups and DXMS group was increased,levels of β-aminohexosidase,histamine,IL-4,IL-6 and TNF-α were decreased,IFN-γ level was increased(P＜0.01),the expressions of p-PI3K/PI3K,p-AKT/AKT,p-p38MAPK/p38MAPK,p-NF-κB/NF-κB and p-ERK/ERK were decreased(P＜0.05,P＜0.01);the number of toluidine blue stained cells in medium,high dose SD extract groups and DXMS group was increased with spindle cell shape,degranulation rate was decreased(P＜0.01).Compared with low dose SD extract group,degranulation rate of high dose SD extract group and DXMS group was decreased(P＜0.01),F-actin expression was increased(P＜0.05),p-p38MAPK/p38MAPK expressions were decreased(P＜0.01).[Conclusion]SD extract inhibited degranulation of IgE sensitized RBL-2H3 cell and decreased the levels of inflammatory mediators,its mechanism may be related to the inhibition the phosphorylation expression of PI3K/AKT,p38MAPK/NF-KB and ERK.

Effect of vascular endothelial growth factor-A165 on scleral remodeling of guinea pigs with form-deprivation myopia / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the effect of the intravitreal injection of vascular endothelial growth factor-A165(VEGF-A165)on the scleral remodeling of guinea pigs with form-deprivation myopia(FDM).METHODS: A total of 120 tricolor guinea pigs, aged three weeks, were randomly divided into 6 groups, with 20 in each group. The blank group did not undergo any intervention. In the FDM group, only the FDM model was established. In the phosphate buffer saline(PBS)group, 2.5 μL of PBS was injected into the vitreous cavity before establishing the FDM model. In the 1ng group, 5ng group, and 10ng group, VEGF-A165 was injected into the vitreous cavity at concentrations of 1, 5 and 10ng, respectively, before the establishment of the FDM model. The FDM model was established by covering the right eyes of guinea pigs with translucent balloons for 14d. The diopter and axial length of the right eyes were measured before and after covering. After 14d, the content of dopamine(DA)in retina was measured by high performance liquid chromatography. Additionally, the mRNA and protein expression levels of matrix metalloproteinase-2(MMP-2), tissue inhibitor of matrix metalloproteinase-2(TIMP-2), transforming growth factor(TGF)-β1, TGF-β2 and α-smooth muscle actin(α-SMA)in sclera were detected by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.RESULTS: Before covering, there were no significant differences in the diopter and axial length of the right eyes of guinea pigs in all groups(P&#x003E;0.05). After 14d of modeling, when compared with the blank group, FDM group showed an increase in the degree of myopia in the right eye, a prolongation of the axial length, a decrease in the content of DA in the retina, and an increase in the expression of MMP-2, TGF-β2 and α-SMA in the sclera. Conversely, the expression of TIMP-2 and TGF-β1 were decreased(P&#x003C;0.01). However, in comparison to the FDM group, the degree of myopia in the 1ng, 5ng, and 10ng groups of guinea pigs decreased, the growth trend of axial length slowed, the content of DA in the retina increased, and the expression of MMP-2, TGF-β2 and α-SMA in the sclera decreased. Furthermore, the expression of TIMP-2 and TGF-β1 in the sclera increased(P&#x003C;0.01). As the concentration of intravitreal injection of VEGF-A165 increased, the degree of myopia in the right eye of guinea pigs gradually increased, and the axial length gradually prolonged. The content of DA in the retina gradually decreased, the expression of MMP-2, TGF-β2, and α-SMA in the sclera gradually increased, while the expression of TIMP-2 and TGF-β1 decreased gradually.CONCLUSION: Intravitreal injection of VEGF-A165 can increase the content of DA in the retina of FDM guinea pigs, affect the expression of MMP-2, TIMP-2, TGF-β1, TGF-β2 and α-SMA in the sclera, and inhibit scleral remodeling of guinea pigs. Notably, the VEGF-A165 at the concentration of 1ng showed the most significant efficacy.

Research progress on the role of macrophages in post-transplantation chronic rejection / 器官移植

Organ transplantation is the optimal treatment for end-stage organ failure. Nevertheless, rejection remains an important factor affecting the allograft survival. At present, acute rejection may be effectively treated, whereas no effective interventions are available for post-transplantation chronic rejection. Long-term chronic rejection may lead to graft failure and severely affect long-term survival rate of allografts. In recent years, the role of macrophages in post-transplantation chronic rejection has gradually captivated increasing attention. In this article, main pathological changes of chronic rejection, the diversity and functional differences of macrophages involved in chronic rejection, and the role and mechanism of macrophages in chronic rejection were reviewed, and research progresses on macrophage-related treatment for chronic rejection were summarized, aiming to provide reference for the study of macrophages in post-transplantation chronic rejection.

Mechanism of rhynchophylline solid lipid nanoparticles inhibiting the proliferation of airway smooth muscle cells in asthmatic model mice / 中国药房

OBJECTIVE To study the inhibitory effect mechanism of rhynchophylline solid lipid nanoparticles （Rhy-SLN） on the proliferation of airway smooth muscle cells （ASMCs） in asthmatic model mice. METHODS Asthma model was prepared by ovalbumin+calmogastrin sensitization. The primary isolation and culture of ASMCs were performed， and morphological observation and identification were also conducted [when α -smooth muscle actin （α -SMA） appeared red and Desmin appeared green in ASMCs， indicating successful cultivation of ASMCs]. The cells were divided into blank group （ASMCs of normal mice）， model group （ASMCs of asthma model mice）， Rhy-SLN group （ASMCs of asthma model mice）， recombinant suppressors of cytokine signaling 1 （SOCS1） overexpression group （ASMCs of asthma model mice transfected with SOCS1 vector）， SOCS1-RNAi group （ASMCs of asthma model mice transfected with SOCS1-RNAi vector） and SB203580 group [p38 mitogen-activated protein kinase （p38 MAPK） inhibitor， ASMCs of asthma model mice]. The cells of each group were added into the corresponding culture medium containing drug （10 μmol/L） or not containing drug for 24 hours. MTT method was used to detect the proliferation of ASMCs in asthmatic mice； Western blot assay was used to detect the protein expressions of α-SMA， interleukin-1β （IL-1β）， SOCS1， p38 MAPK and phosphorylated p38 MAPK （p-p38 MAPK） in ASMCs. RESULTS The primary ASMCs of mice varied in shape and size， presenting irregular， spindle and triangular shapes；α-SMA appeared red and Desmin appeared green， indicating successful cultivation of ASMCs. Compared with model group， ASMCs absorbance values and protein expressions of α -SMA， p38 MAPK， and p-p38 MAPK were reduced significantly in Rhy- SLN group， SOCS1 overexpression group and SB203580 E-mail：wangmeng106@163.com group， while protein expression of SOCS1 （except for group） was increased significantly （P＜0.05）； protein expressions of IL-1β was reduced significantly in ASMCs （P＜ 0.05）. ASMCs absorbance values and protein expressions of α-SMA， SOCS1， p38 MAPK and p-p38 MAPK were increased significantly in SOCS1-RNAi group （P＜0.05）. CONCLUSIONS Rhy-SLN can inhibit the proliferation of ASMCs， the mechanism of which may be associated with overexpression of SOCS1 and inhibiting the protein expressions of IL-1β and p38 MAPK.

Effects of acupuncture on the lung tissue of asthma model rats of expression levels of α-smooth muscle actin and fibronectin / 中国中西医结合急救杂志

Objective To observe the effect of acupuncture on lung tissue of asthma model rats ofα-smooth muscle actin(α-SMA),fibronectin(FN)expression levels.Methods A total of 40 male SPF Sprague-Dawley(SD)rats were randomly divided into normal control group,model group,drug group and acupuncture group,with 10 rats in each group.On the 1st and 8th days of the experiment,the normal control group was intraperitoneally injected with 1 mL of 0.9％ sodium chloride solution,and the model group was intraperitoneally injected with 1 mL of 10％ compound ovalbumin solution to sensitize.On the 15th day of the experiment,the normal control group was nebulized with 0.9％ sodium chloride solution for 20 minutes,and the model group was nebulized with 1％ ovalbumin solution for 20 minutes to stimulate asthma.After successful modeling,the normal control group and the model group were not treated,and the drug group was intraperitoneally injected with 0.05％ dexamethasone sodium phosphate solution,once a day,for 10 days.In the acupuncture group,Feishu,Dazhui and Fengmen acupoints were punctured once every 2 days for 7 times.The mRNA expression of signal transducer and activator of transcription 6(STAT6),the protein and positive expression of transforming growth factor-β1(TGF-β1),and the levels ofα-SMA,FN and peripheral blood T lymphocyte populations(CD3+,CD4+,CD8+)in the lung tissues of the four groups were compared.Results Compared with the normal control group,STAT6 mRNA expression,TGF-β1 protein and positive expression,the positive expression ofα-SMA,FN and T lymphocyte populations(CD3+,CD8+)in peripheral blood in model group were significantly increased[STAT6 mRNA(2-ΔΔCt):13.12±2.20 vs.1.02±0.20,TGF-β1 protein expression(A value):0.10±0.01 vs.0.06±0.01,TGF-β1 positive expression(A value):0.11±0.01 vs.0.08±0.01,α-SMA positive expression(A value):19.65±1.37 vs.7.74±0.81,FN positive expression(A value):18.40±0.79 vs.6.50±0.60,CD3+:0.70±0.03 vs.0.59±0.02,CD8+:0.39±0.02 vs.0.20±0.02,all P<0.05],and CD4+in model group was significantly decreased(0.32±0.02 vs.0.39±0.03,P<0.05);STAT6 mRNA expression and TGF-β1 protein expression,the positive expression ofα-SMA and FN and T lymphocyte populations(CD3+,CD8+)in peripheral blood in the drug group and acupuncture group were significantly lower than those in the model group,CD4+in the drug group and acupuncture group was significantly higher than that in the model group,and the changes in the acupuncture group was more significant than those in the drug group[STAT6 mRNA(2-ΔΔCt):3.01±0.55 vs.5.64±0.65,TGF-β1 protein expression(A value):0.06±0.01 vs.0.09±0.01,TGF-β1 positive expression(A value):0.08±0.01 vs.0.09±0.01,α-SMA positive expression(A value):12.33±0.50 vs.14.99±0.53,FN positive expression(A value):9.91±0.61 vs.13.19±0.66,CD3+:0.60±0.03 vs.0.65±0.04,CD4+:0.39±0.02 vs.0.36±0.02,CD8+:0.21±0.04 vs.0.28±0.03,all P<0.05].Conclusion For asthma model rats,acupuncture at Feishu,Dazhui and Fengmen acupoints can significantly stimulate the nerves and muscles of lung tissue,promote blood circulation in the lung,enhance lung function,improve the health status of lung tissue,reduce the content of the positive expression ofα-SMA and FN,and improve airway remodeling.

Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii / 中国中药杂志

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

CXCR5 Regulates Neuronal Polarity Development and Migration in the Embryonic Stage via F-Actin Homeostasis and Results in Epilepsy-Related Behavior / 神经科学通报·英文版

Epilepsy is a common, chronic neurological disorder that has been associated with impaired neurodevelopment and immunity. The chemokine receptor CXCR5 is involved in seizures via an unknown mechanism. Here, we first determined the expression pattern and distribution of the CXCR5 gene in the mouse brain during different stages of development and the brain tissue of patients with epilepsy. Subsequently, we found that the knockdown of CXCR5 increased the susceptibility of mice to pentylenetetrazol- and kainic acid-induced seizures, whereas CXCR5 overexpression had the opposite effect. CXCR5 knockdown in mouse embryos via viral vector electrotransfer negatively influenced the motility and multipolar-to-bipolar transition of migratory neurons. Using a human-derived induced an in vitro multipotential stem cell neurodevelopmental model, we determined that CXCR5 regulates neuronal migration and polarization by stabilizing the actin cytoskeleton during various stages of neurodevelopment. Electrophysiological experiments demonstrated that the knockdown of CXCR5 induced neuronal hyperexcitability, resulting in an increased number of seizures. Finally, our results suggested that CXCR5 deficiency triggers seizure-related electrical activity through a previously unknown mechanism, namely, the disruption of neuronal polarity.

Effect of mycophenolate mofetil alleviates carbon tetrachloride-induced liver fibrosis in mice / 中南大学学报(医学版)

OBJECTIVES@#Hepatic fibrosis is a serious pathological consequence of chronic liver disease. Mycophenolate mofetil (MMF) is a commonly used immunosuppressant after organ transplant. However, the relationship between MMF and hepatic fibrosis remains unclear. This study aims to explore the effect of MMF on hepatic fibrosis in mice and the potential mechanism.@*METHODS@#A total of 24 mice (male, 8-week old, C57BL/6) were randomly divided into a control group, a MMF group, a carbon tetrachloride (CCl4) group and a CCl4+MMF group (n=6 in each group). After the mice were sacrificed, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected. The liver tissues were taken up for Masson staining and collagen I (COL1) immunohistochemistry. The levels of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) were detected by Western blotting. Finally, the levels of mRNA for TGF-β1, α-SMA, and COL1 were detected using real-time PCR.@*RESULTS@#Compared with the CCl4 group, the ALT and AST levels were lower (both P<0.05), the degree of liver fibrosis was alleviated, and the deposition of COL1 in the liver was significantly decreased (P<0.01) in the CCl4+MMF group. Compared with the CCl4 group, the protein expression levels of TGF-β1 and α-SMA were significantly decreased (both P<0.05) and the relative expression levels of TGF-β1, α-SMA and COL1 mRNA in the liver were significantly decreased (all P<0.05) in the CCl4+MMF.@*CONCLUSIONS@#MMF could reduce CCl4-induced hepatic fibrosis, which might be related to the inhibition of TGF-β1. This study is expected to provide a target for the treatment of hepatic fibrosis.

Gelsolin inhibits autophagy by regulating actin depolymerization in pancreatic ductal epithelial cells in acute pancreatitis

Gelsolin (GSN) can sever actin filaments associated with autophagy. This study investigated how GSN-regulated actin filaments control autophagy in pancreatic ductal epithelial cells (PDECs) in acute pancreatitis (AP). AP was produced in a rat model and PDECs using caerulein (CAE). Rat pancreatic duct tissue and HPDE6-C7 cells were extracted at 6, 12, 24, and 48 h after CAE treatment. HPDE6-C7 cells in the presence of CAE were treated with cytochalasin B (CB) or silenced for GSN for 24 h. Pancreatic histopathology and serum amylase levels were analyzed. Cellular ultrastructure and autophagy in PDECs were observed by transmission electron microscopy after 24 h of CAE treatment. The expression of GSN and autophagy markers LC3, P62, and LAMP2 was evaluated in PDECs by immunohistochemistry and western blotting. Actin filaments were observed microscopically. Amylase levels were highest at 6 h of AP, and pancreatic tissue damage increased over time. Mitochondrial vacuolization and autophagy were observed in PDECs. CAE increased GSN expression in these cells over time, increased the LC3-II/LC3-I ratio and LAMP2 expression at 24 and 6 h of treatment, respectively, and decreased P62 expression at all time points. CB treatment for 24 h decreased the LC3-II/LC3-I ratio and LAMP2 expression, increased P62 levels, but had no impact on GSN expression in CAE-treated PDECs. CAE induced actin depolymerization, and CB potentiated this effect. GSN silencing increased the LC3-II/LC3-I ratio and LAMP2 expression and reduced actin depolymerization in CAE-treated PDECs. GSN may inhibit autophagosome biogenesis and autophagosome-lysosome fusion by increasing actin depolymerization in PDECs in AP.

Plasma exosomal microRNAs are non-invasive biomarkers of moyamoya disease: A pilot study

Abstract Background As a progressive cerebrovascular disease, Moyamoya Disease (MMD) is a common cause of stroke in children and adults. However, the early biomarkers and pathogenesis of MMD remain poorly understood. Methods and material This study was conducted using plasma exosome samples from MMD patients. Next-generation high-throughput sequencing, real-time quantitative PCR, gene ontology analysis, and Kyoto Encyclopaedia of Genes and Genomes pathway analysis of ideal exosomal miRNAs that could be used as potential biomarkers of MMD were performed. The area under the Receiver Operating Characteristic (ROC) curve was used to evaluate the sensitivity and specificity of biomarkers for predicting events. Results Exosomes were successfully isolated and miRNA-sequence analysis yielded 1,002 differentially expressed miRNAs. Functional analysis revealed that they were mainly enriched in axon guidance, regulation of the actin cytoskeleton and the MAPK signaling pathway. Furthermore, 10 miRNAs (miR-1306-5p, miR-196b-5p, miR-19a-3p, miR-22-3p, miR-320b, miR-34a-5p, miR-485-3p, miR-489-3p, miR-501-3p, and miR-487-3p) were found to be associated with the most sensitive and specific pathways for MMD prediction. Conclusions Several plasma secretory miRNAs closely related to the development of MMD have been identified, which can be used as biomarkers of MMD and contribute to differentiating MMD from non-MMD patients before digital subtraction angiography.

Cloning the full-length CDNA of actin gene and analysing alliinase gene expression in tillering onion

Tillering onion is a herbaceous plant belonging to the Liliaceae family. We cloned the cDNAs of the actin gene (AcACT, GenBank: MF919598) of tillering onion using rapid amplification of the cDNA ends. The full-length cDNA of AcACT was 1,357 bp long with an open reading frame of 1,131 bp encoding 376 amino acids. The amino acid sequence of AcACT shared > 96% similarity with the amino acid sequences of other ACTs and was found (by means of phylogenetic tree analysis) to be closely related to those of Ananas comosus and Papaver somniferum. AcACT expressions showed no significant differences (p > 0.01) in two cultivars L-SH and L-SY over three growth periods and under suitable conditions, low temperature, and short-day conditions. In addition, AcACT was used as an internal reference gene to analyse the expression of the alliinase gene (AcALL). AcALL expression trends in the roots, stems and leaves were consistent with those of diallyl disulphide and diallyl trisulphide. Thus, AcACT is highly conserved and can be used as a suitable internal reference gene when analysing gene expression in tillering onion.

Primary leiomyosarcoma of ovary: A rare malignancy as an incidental finding

Primary leiomyosarcoma (PLMS) of the ovary is extremely rare tumors comprising 1% of ovarian tumors. About 3% of all ovarian malignancies are primary ovarian sarcomas. Only 72 cases have been reported till date. A 57-year-old postmenopausal female presented with abdominal pain for the last 6 months. Ultrasonography and MRI revealed a heterogeneously enhancing solid lobulated mass in the left adnexa abutting the fundus of the uterus and bowel loops. The endometrial cavity was normal. Ovarian markers CA 125, CEA, CA 19.9, and all hematological parameters were within normal limits. LDH was near normal (284 IU/ml). The specimen was sent for frozen section and a diagnosis of malignant spindle cell lesion of ovary was rendered. Histopathology of the ovarian mass revealed intersecting fascicles of tumor cells consisting of ovoid to spindle-shaped cells having a moderate amount of cytoplasm. Bizarre and atypical cells were seen singly dispersed and in small aggregates along with the brisk mitotic activity. Focal areas of necrosis and hemorrhage were also noted. Immunohistochemistry showed strong positivity for smooth muscle actin and Caldesmon while focal positivity for Desmin and Epithelial Membrane Antigen (EMA) was noted. The lesion was negative for Inhibin, Calretinin, and CD 117 and S100. The final diagnosis of primary ovarian Leiomyosarcoma was given based on histopathology and Immunohistochemistry. PLMS of the ovary are rare incidental findings in postmenopausal women. These are highly malignant tumors and carry a poor prognosis. Hence, early diagnosis and surgical treatment with cytoreduction improve patient survival.

Arracacia xanthorrhiza Bancr compounds decrease β-actin, hypoxia-inducible factor-1 and nitric oxide production in HeLa cells / Los componentes de la Arracacia xanthorrhiza Bancr disminuyen las producciones de la actina β, del factor-1 inducible por la hipoxia y del óxido nítrico en células Hela

Abstract The treatment of cancer patients with anti-cancer drugs is often accompanied by the presence of undesirable side effects. The use of natural plant derivatives alone, or in conjunction with existing anti-neoplastic drugs, has been suggested to obtain better results and decrease these side effects. Nitric oxide (NO•), the hypoxia-inducible factor-1 (HIF-1), and decreased concentration of actin play important roles in cancer progression. The beneficial effects of polyphenols in various organ disorders including cancer has been reported. The aim of this study was to determine the effect of Arracacia xanthorrhiza Bancr extracts, white (WAXB) and red (RAXB) variants (compounds rich in polyphenols) on the concentrations of β-actin, NO• and HIF-1 in Hela cells cultures, to uncover possible anti-neoplastic effects. Extracts from the plant leaves were added to Hela cell cultures at a concentration of 10-3 mg/mL, and after 24 hours of culture, the concentrations of β-actin, NO• and HIF-1 were determined by immunohistochemical, biochemical and western blot assays. Both extracts reduced the concentrations of β-actin, NO• and HIF-1 (p<0.001), similar to the methotrexate effect. These results suggest an antineoplastic effect of the studied plant extracts and highlight the possibility of their use in the treatment of neoplasms.

Resumen El tratamiento de pacientes con cáncer utilizando drogas-antineoplásicas presenta problemas relacionados con efectos colaterales indeseables. Se ha sugerido el uso de derivados de plantas naturales solas, o en combinación con drogas antineoplásicas existentes para obtener mejores resultados y disminuir los efectos colaterales. Así mismo, se ha reportado que el óxido nítrico (NO•), el factor-1 inducible por hipoxia (HIF-1) y la disminución de la expresión de la actina tienen un papel en la progresión del cáncer. También se ha reportado los efectos beneficiosos de lo polifenoles en varios desordenes orgánicos, incluyendo el cáncer. El objetivo de este estudio fue determinar los efectos de los extractos procedentes de la Arracacia xanthorrhiza Bancr blanca (AXBB) y la variedad roja (AXBR) (compuestos ricos en polifenoles) en las concentraciones de la actina-beta, el NO• y el HIF-1 en cultivo de células Hela, para destacar sus posibles efectos antineoplásicos. A los cultivos de células Hela se les agregaron los extractos de las hojas de AXBB o AXBR (10-3 mg/mL, concentración final) y después de 24 horas de cultivo se determinaron las concentraciones de la actina-beta, el NO• y el HIF-1 por métodos inmunohistoquímicos, bioquímicos y western blot. Ambos extractos disminuyeron las concentraciones de la actina-beta, el NO• y el HIF-1 (p<0,001) de una manera similar al efecto del metotrexato. Estos resultados sugieren un efecto antineoplásico de estos extractos y destacan la posibilidad de ser usados para el tratamiento de las neoplasias.

Yes-associated protein ameliorates tumor necrosis factor-α-induced epithelial barrier dysfunction by affecting the F-actin / 中国小儿急救医学

Objective:To investigate the protective role of Yes-associated protein(YAP)in intestinal epithelial barrier injury.Methods:The intestinal epithelial barrier model was established by culturing human colorectal adenocarcinoma cell line Caco-2 cells, which were divided into four groups: control group, Caco-2 monolayers did not receive any treatment; recombinant human tumor necrosis factor-α(rhTNF-α)group, 100 μg/L of rhTNF-α was added to Caco-2 monolayers; vector+ rhTNF-α group, Caco-2 monolayers were first added with control plasmid pcDNA3.1-vector, and 100 μg/L rhTNF-α was added 24 hours later; YAP+ rhTNF-α group, Caco-2 cells with barrier construction were first added with pcDNA3.1-YAP, and 100 μg/L rhTNF-α was added 24 hours later.Realtime-PCR and Western blot were used to evaluate YAP mRNA and protein expression level.Epithelial permeability was assayed by trans-epithelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran 40(FD-40 flu). Cellular distribution of F-actin was assayed by immunofluorescence staining.Results:Compared with control group[(607.3±29.3)Ω·cm 2], TEER of rhTNF-α group[(265.3±32.7)Ω·cm 2] decreased, while TEER of YAP+ rhTNF-α group[(387.0±18.7)Ω·cm 2]increased compared with rhTNF-α group, the differences were statistically significant( P<0.001). The FD-40 flux of rhTNF-α(22.7%±0.5%) group was higher than that of the control group(6.3%±0.9%), while the FD-40 flux of Yap + rhTNF-α group(12.2%±0.8%) was lower than that of rhTNF-α group, the differences were statistically significant( P<0.001). Immunofluorescence staining showed that compared with the control group, the cytoskeletal F-actin fiber dense spot decreased in rhTNF-α group, and some cells showed obvious trans-cellular stress fiber structure, while the peripheral actin band was clear in YAP+ rhTNF-α group, and the intracellular stress fiber decreased.YAP+ TNF-α group appeared as a clear, peripheral actin ribbon with a decrease in cytoplasmic stress fibres. Conclusion:YAP overexpression significantly inhibits TNF-α induced decline of TEER, and increases of FD-40 flux and F-actin rearrangement of Caco-2.YAP could ameliorate TNF-α induced intestinal epithelial barrier injury by regulating cytoskeleton F-actin.

Effect of Morindae Officinalis Radix Processed with Different Proportions of Glycyrrhizae Radix et Rhizoma on Improvement of Renal Function and HPG Axis of Kidney Yang Deficiency Model Rats Induced by Adenine / 中国实验方剂学杂志

ObjectiveTo explain the scientific connotation of Morindae Officinalis Radix （MOR） processed by Glycyrrhizae Radix et Rhizoma （Gly） by comparing the effect of raw products of MOR and processed products of MOR with different proportions of Gly （GMOs） on the improvement of renal function and hypothalamic-pituitary-gonadal （HPG） axis， the protein expression of Wnt/β-catenin and transforming growth factor-β1 （TGF-β1）/Smad signal pathways in kidney Yang deficiency model rats induced by adenine. MethodGMOs were prepared according to method under MOR in 2020 edition of Chinese Pharmacopoeia. Rat model of kidney Yang deficiency was established by intragastrical administration of adenine， levels of follicle stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E2） and testosterone （T） were measured by enzyme-linked immunosorbent assay （ELISA）. Levels of urea nitrogen （BUN） and serum creatinine （SCr） were measured by spectrophotometry， hematoxylin-eosin （HE） staining was used to evaluate the pathological changes of kidney， testis and epididymis. Immunohistochemistry （IHC） was used to analyze the protein expression of E-cadherin， α-smooth muscle actin （α-SMA）， Wnt2b， β-catenin， Smad1 and Smad4. ResultMOR processed with 100∶6 and 100∶12 proportions of Gly （short for GMO/100∶6 and GMO/100∶12） had the most obvious improvement on the body posture of kidney Yang deficiency model rats. GMO/100∶12 had the best effect on reducing the levels of BUN， SCr， FSH， LH and the ratio of E2/T. GMO/100∶6 and GMO/100∶12 had the best effect on regulating the protein expression of E-cadherin， α-SMA， Wnt2b， β-catenin， Smad1 and Smad4. ConclusionGMO/100∶6 and GMO/100∶12 have the a good effect on the improvement of renal function and HPG axis in kidney Yang deficiency model rats induced by adenine， which is related with the fact that they can regulate Wnt/β-catenin pathway in renal and testicular tissue and TGF-β1/Smads pathway in testicular tissue.

Effect of Morindae Officinalis Radix Processed with Different Proportions of Glycyrrhizae Radix et Rhizoma on Improvement of Renal Function and HPG Axis of Kidney Yang Deficiency Model Rats Induced by Adenine / 中国实验方剂学杂志

ObjectiveTo explain the scientific connotation of Morindae Officinalis Radix （MOR） processed by Glycyrrhizae Radix et Rhizoma （Gly） by comparing the effect of raw products of MOR and processed products of MOR with different proportions of Gly （GMOs） on the improvement of renal function and hypothalamic-pituitary-gonadal （HPG） axis， the protein expression of Wnt/β-catenin and transforming growth factor-β1 （TGF-β1）/Smad signal pathways in kidney Yang deficiency model rats induced by adenine. MethodGMOs were prepared according to method under MOR in 2020 edition of Chinese Pharmacopoeia. Rat model of kidney Yang deficiency was established by intragastrical administration of adenine， levels of follicle stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E2） and testosterone （T） were measured by enzyme-linked immunosorbent assay （ELISA）. Levels of urea nitrogen （BUN） and serum creatinine （SCr） were measured by spectrophotometry， hematoxylin-eosin （HE） staining was used to evaluate the pathological changes of kidney， testis and epididymis. Immunohistochemistry （IHC） was used to analyze the protein expression of E-cadherin， α-smooth muscle actin （α-SMA）， Wnt2b， β-catenin， Smad1 and Smad4. ResultMOR processed with 100∶6 and 100∶12 proportions of Gly （short for GMO/100∶6 and GMO/100∶12） had the most obvious improvement on the body posture of kidney Yang deficiency model rats. GMO/100∶12 had the best effect on reducing the levels of BUN， SCr， FSH， LH and the ratio of E2/T. GMO/100∶6 and GMO/100∶12 had the best effect on regulating the protein expression of E-cadherin， α-SMA， Wnt2b， β-catenin， Smad1 and Smad4. ConclusionGMO/100∶6 and GMO/100∶12 have the a good effect on the improvement of renal function and HPG axis in kidney Yang deficiency model rats induced by adenine， which is related with the fact that they can regulate Wnt/β-catenin pathway in renal and testicular tissue and TGF-β1/Smads pathway in testicular tissue.

Role of three drugs in traumatic proliferative vitreoretinopathy in rabbits / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effect of triamcinolone acetonide(TA), artesunate(ART), and luteolin(LU)on the prevention and treatment of traumatic proliferative vitreoretinopathy(TPVR). <p>METHODS: Forty-eight cyanotic blue rabbits were selected to prepare TPVR animal models by making a penetrating eye injury and intravitreal injection of 0.3mL platelet-rich plasma, and were randomly divided into four groups(<i>n</i>=12), in which the vitreous cavity of the control group was injected with 0.1mL saline; The vitreous cavity of the TA group was injected with 0.1mL(1mg/mL)triamcinolone acetonide; The vitreous cavity of the ART group was injected with 0.1mL(20μg/mL)artesunate; 0.1mL(10μg/mL)luteolin was injected into the vitreous cavity of the LU group. The vitreous and retinal proliferation were observed by fundus photography and ocular ultrasound at 1, 2, 3 and 4wk postoperatively. The expression levels of α-SMA and VIM protein in the vitreous fluid of each group of rabbit eyes were detected by Western Blot at 28d postoperatively, and the retinal tissue structure of each group was observed by retinal HE staining. <p>RESULTS: At 28d postoperatively, the TPVR grading of rabbit eyes in the TA, ART and LU groups were significantly lower than that in the control group(<i>P</i><0.05), and the TPVR grading of rabbit eyes in the TA group was significantly lower than that in the ART and LU groups(<i>P</i><0.05). The expression levels of α-SMA and VIM proteins in the vitreous fluid of the rabbit eyes in the TA, ART and LU groups were significantly lower than those in the control group at 28d after surgery(<i>P</i><0.01). The results of HE staining showed that the arrangement of retinal layers in rabbit eyesin the control group were disordered, severely distorted or locally broken, the structure of each layer were unclear, the anterior membrane was obviously thickened, and the retina was obviously detached; The arrangement of retinal layersin rabbit eyes in the LU group were slightly distorted, inflammatory exudation was visible in front of the retina, and the retina was superficially detached; The structure of retina in rabbit eyes in the ART group were clear, with mild edema and superficial detachment; The structure of retinal layers in rabbit eyes in the TA group were clear, the arrangement was still neat, the retinal folds were locally visible, and there was no retinal detachment.<p>CONCLUSION: Intravitreal injection of triamcinolone acetonide, artesunate and luteolin were all effective in preventing and treating traumatic TPVR, among which triamcinolone acetonide has the most obvious effect.