Huangqi Decoction, a compound Chinese herbal medicine, inhibits the proliferation and activation of hepatic stellate cells by regulating the long noncoding RNA-C18orf26-1/microRNA-663a/transforming growth factor-β axis / 中西医结合学报

OBJECTIVE@#Huangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis.@*METHODS@#The expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting.@*RESULTS@#Lnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-β1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-β1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-β1, TGF-β type I receptor (TGF-βRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment.@*CONCLUSION@#Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.

Inducible co-stimulatory molecules participate in mesenteric vascular endothelial-mesenchymal transition and sclerosis of mesenteric vessels in spontaneously hypertensive rats / 南方医科大学学报

OBJECTIVE@#To investigate the correlation of inducible co-stimulatory molecules (ICOS) with mesenteric vascular endothelial- mesenchymal transition (EndMT) and sclerosis in spontaneously hypertensive rats (SHR).@*METHODS@#Twenty 4-week-old WKY rats and 20 SHRs of the same strain were both randomly divided into 4 groups for observation at 4, 6, 10 and 30 weeks of age. ICOS expression frequency in rat spleen CD4+T cells was analyzed using flow cytometry, and the expressions of ICOS, VE-cad, α-SMA and Col3 mRNA in rat mesentery were detected by RT-PCR. The distributions of ICOS, IL-17A and TGF-β in rat mesentery were detected by immunohistochemistry. The levels of IL-17A and TGF-β in rat plasma were measured using ELISA. The morphological changes of rat mesenteric vessels were observed with Masson staining. Spearman or Pearson correlation analyses were used to evaluate the correlation between ICOS expression and the expressions of the markers of vascular EndMT and sclerosis.@*RESULTS@#Compared with the control WKY rats, the SHRs began to show significantly increased systolic blood pressure and ICOS expression frequency on CD4+T cells at 6 weeks of age (P < 0.05). In the SHRs, the mRNA and protein expressions of ICOS, α-SMA, Col3, IL-17A and TGF-β in the mesentery were significantly higher than those in control group (P < 0.05), while the mRNA and protein expressions of VE-cad started to reduce significantly at 10 weeks of age (P < 0.05). The plasma levels of IL-17A and TGF-β were significantly increased in SHRs since 6 weeks of age (P < 0.05) with progressive worsening of mesenteric vascular sclerosis (P < 0.05). ICOS mRNA and protein expression levels in the mesenteric tissues of SHRs began to show positive correlations with α-SMA and Col3 expression levels and the severity of vascular sclerosis at 6 weeks of age (P < 0.05) and a negative correlation with VE-cad expression level at 10 weeks (P < 0.05).@*CONCLUSION@#ICOS play an important pathogenic role in EndMT and sclerosis of mesenteric vessels in essential hypertension by mediating related immune responses.

Effect of recombinant human fibroblast growth factor 21 on the mineralization of cementoblasts and its related mechanism / 华西口腔医学杂志

OBJECTIVES@#To investigate the effect of recombinant human fibroblast growth factor 21 (rhFGF21) on the proliferation and mineralization of cementoblasts and its mechanism.@*METHODS@#Hematoxylin eosin, immunohistochemical staining, and immunofluorescence were used to detect the expression and distribution of fibroblast growth factor 21 (FGF21) in rat periodontal tissues and cementoblasts (OCCM-30), separately. Cell Counting Kit-8 was used to detect the proliferation of OCCM-30 under treatment with rhFGF21. Alkaline phosphatase staining and Alizarin Red staining were used to detect the mineralization state of OCCM-30 after 3 and 7 days of mineralization induction. The transcription and protein expression of the osteogenic-related genes Runx2 and Osterix were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot analysis. The expression levels of genes of transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling pathway in OCCM-30 were detected through PCR array analysis.@*RESULTS@#FGF21 was expressed in rat periodontal tissues and OCCM-30. Although rhFGF21 had no significant effect on the proliferation of OCCM-30, treatment with 50 ng/mL rhFGF21 could promote the mineralization of OCCM-30 cells after 7 days of mineralization induction. The transcriptional levels of Runx2 and Osterix increased significantly at 3 days of mineralization induction and decreased at 5 days of mineralization induction. Western blot analysis showed that the protein expression levels of Runx2 and Osterix increased during mineralization induction. rhFGF21 up-regulated Bmpr1b protein expression in cells.@*CONCLUSIONS@#rhFGF21 can promote the mineralization ability of OCCM-30. This effect is related to the activation of the TGFβ/BMP signaling pathway.

Regulation of colony-stimulating factor 1 receptor inhibitor pexidartinib on the senescence of mouse bone marrow-derived macrophages stimulated by lipopolysaccharide / 中华口腔医学杂志

Objective: To investigate the effects of colony-stimulating factor 1 receptor (CSF-1R) inhibitor pexidartinib (PLX3397) on the senescence of bone marrow-derived macrophages (BMDM) stimulated by lipopolysaccharide (LPS). Methods: BMDM were isolated and cultured from femurs and tibiae of 10 male C57BL/6 mice aged 6-8 weeks (obtained from Laboratory Animal Center of Guizhou Medical University). They were divided into blank control group, LPS group (treated with 1 μg/ml LPS for 24 h) as well as low, medium and high concentration PLX3397 pretreatment groups (treated with 100, 500 and 1 000 nmol/L PLX3397 for 4 h respectively followed by 1 μg/ml LPS for 24 h). The corresponding markers of macrophages were detected by flow cytometry. Cell viability was detected by cell counting kit-8 and cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-gal) staining. Meanwhile, protein expressions of cycle-dependent kinase inhibitor p16, p21 and CSF-1R were detected by Western blotting, and the expressions of p16 and p21 were detected by intracellular immunofluorescence. Real-time fluorescence quantitative PCR (RT-qPCR) was used to investigate the mRNA levels of senescence-associated secretory phenotype (SASP) genes including interleukin (IL), IL-1β, chemokine-1/10 (CXCL-1/10), matrix metalloproteinase-8 (MMP-8), and transforming growth factor-β (TGF-β). Results: The rate of SA-β-gal positive staining in medium and high concentration PLX3397 pretreatment groups [(39.33±4.93)% and (36.33±3.06)% respectively] were significantly downregulated compared with LPS group [(52.00±3.00)%] (P=0.020, P=0.005). The expression of CSF-1R protein in low, medium and high concentration PLX3397 pretreatment groups were (0.74±0.18, 0.61±0.07, 0.54±0.06), all of which were significantly lower than that in LPS group (1.16±0.08) (P=0.013, P=0.002, P<0.001). The expression levels of CSF-1R mRNA in low, medium and high concentration PLX3397 pretreatment groups (1.04±0.06, 0.90±0.05, 1.18±0.08) showed similar trend (2.90±0.25) (P<0.001). The average fluorescence intensity of p16 in all PLX3397 pretreatment groups were 49.76±3.65, 48.21±1.72, 47.99±1.26 respectively, which were significantly lower than that in LPS group (66.88±5.85) (P=0.001, P<0.001, P<0.001). The average fluorescence intensity of p21 in medium and high concentration PLX3397 pretreatment groups were (34.43±3.62, 30.13±0.86), significantly lower than that in LPS group (46.82±5.33) (P=0.043, P=0.007). The expression of p16 protein in low, medium and high concentration PLX3397 pretreatment groups (0.56±0.04, 0.55±0.04, 0.35±0.19) were significantly lower than that in LPS group (0.98±0.10) (P=0.003, P=0.002, P<0.001), as well the expression of p21 protein (0.69±0.20, 0.42±0.08, 0.26±0.14) (P=0.032, P=0.002, P<0.001). According to the results of RT-qPCR, the expressions of IL-6, IL-1β, CXCL-1, CXCL-10 and MMP-8 in PLX3397 pretreatment groups were significantly lower than those in LPS group (P<0.001), while the expression of TGF-β increased (P<0.001). Conclusions: LPS could induce the cell senescence, increase the secretion of SASP and aggravate local inflammation by activating the CSF-1R on the cell surface of bone marrow-derived macrophages. CSF-1R inhibitor PLX3397 might attenuate CSF-1R activation associated with LPS and inhibit the senescence of bone marrow-derived macrophages induced by LPS.

Influence of autologous adipose stem cell matrix gel on wound healing and scar hyperplasia of full-thickness skin defects in rabbit ears / 中华烧伤杂志

Objective: To investigate the influence of autologous adipose stem cell matrix gel on wound healing and scar hyperplasia of full-thickness skin defects in rabbit ears, and to analyze the related mechanism. Methods: Experimental research methods were adopted. The complete fat pads on the back of 42 male New Zealand white rabbits aged 2 to 3 months were cut to prepare adipose stem cell matrix gel, and a full-thickness skin defect wound was established on the ventral side of each ear of each rabbit. The left ear wounds were included in adipose stem cell matrix gel group (hereinafter referred to as matrix gel group), and the right ear wounds were included in phosphate buffer solution (PBS) group, which were injected with autologous adipose stem cell matrix gel and PBS, respectively. The wound healing rate was calculated on post injury day (PID) 7, 14, and 21, and the Vancouver scar scale (VSS) scoring of scar tissue formed on the wound (hereinafter referred to as scar tissue) was performed in post wound healing month (PWHM) 1, 2, 3, and 4. Hematoxylin-eosin staining was performed to observe and measure the histopathological changes of wound on PID 7, 14, and 21 and the dermal thickness of scar tissue in PWHM 1, 2, 3, and 4. Masson staining was performed to observe the collagen distribution in wound tissue on PID 7, 14, and 21 and scar tissue in PWHM 1, 2, 3, and 4, and the collagen volume fraction (CVF) was calculated. The microvessel count (MVC) in wound tissue on PID 7, 14, and 21 and the expressions of transforming growth factor β1 (TGF-β1) and α smooth muscle actin (α-SMA) in scar tissue in PWHM 1, 2, 3, and 4 were detected by immunohistochemical method, and the correlation between the expression of α-SMA and that of TGF-β1 in scar tissue in matrix gel group was analyzed. The expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) in wound tissue were detected by enzyme-linked immunosorbent assay on PID 7, 14, and 21. The number of samples at each time point in each group was 6. Data were statistically analyzed with analysis of variance for repeated measurement, analysis of variance for factorial design, paired sample t test, least significant difference test, and Pearson correlation analysis. Results: On PID 7, the wound healing rate in matrix gel group was (10.3±1.7)%, which was close to (8.5±2.1)% in PBS group (P>0.05). On PID 14 and 21, the wound healing rates in matrix gel group were (75.5±7.0)% and (98.7±0.8)%, respectively, which were significantly higher than (52.7±6.7)% and (90.5±1.7)% in PBS group (with t values of 5.79 and 10.37, respectively, P<0.05). In PWHM 1, 2, 3, and 4, the VSS score of scar tissue in matrix gel group was significantly lower than that in PBS group (with t values of -5.00, -2.86, -3.31, and -4.45, respectively, P<0.05). Compared with the previous time point within the group, the VSS score of scar tissue at each time point after wound healing in the two groups was significantly increased (P<0.05), except for PWHM 4 in matrix gel group (P>0.05). On PID 7, the granulation tissue regeneration and epithelialization degree of the wounds between the two groups were similar. On PID 14 and 21, the numbers of fibroblasts, capillaries, and epithelial cell layers in wound tissue of matrix gel group were significantly more than those in PBS group. In PWHM 1, 2, 3, and 4, the dermal thickness of scar tissue in matrix gel group was significantly thinner than that in PBS group (with t values of -4.08, -5.52, -6.18, and -6.30, respectively, P<0.05). Compared with the previous time point within the group, the dermal thickness of scar tissue in the two groups thickened significantly at each time point after wound healing (P<0.05). Compared with those in PBS group, the collagen distribution in wound tissue in matrix gel group was more regular and the CVF was significantly increased on PID 14 and 21 (with t values of 3.98 and 3.19, respectively, P<0.05), and the collagen distribution in scar tissue was also more regular in PWHM 1, 2, 3, and 4, but the CVF was significantly decreased (with t values of -7.38, -4.20, -4.10, and -4.65, respectively, P<0.05). Compared with the previous time point within the group, the CVFs in wound tissue at each time point after injury and scar tissue at each time point after wound healing in the two groups were significantly increased (P<0.05), except for PWHM 1 in matrix gel group (P>0.05). On PID 14 and 21, the MVC in wound tissue in matrix gel group was significantly higher than that in PBS group (with t values of 4.33 and 10.10, respectively, P<0.05). Compared with the previous time point within the group, the MVC of wound at each time point after injury in the two groups was increased significantly (P<0.05), except for PID 21 in PBS group (P>0.05). In PWHM 1, 2, 3, and 4, the expressions of TGF-β1 and α-SMA in scar tissue in matrix gel group were significantly lower than those in PBS group (with t values of -2.83, -5.46, -5.61, -8.63, -10.11, -5.79, -8.08, and -11.96, respectively, P<0.05). Compared with the previous time point within the group, the expressions of TGF-β1 and α-SMA in scar tissue in the two groups were increased significantly at each time point after wound healing (P<0.05), except for the α-SMA expression in matrix gel group in PWHM 4 (P>0.05). There was a significantly positive correlation between the expression of α-SMA and that of TGF-β1 in scar tissue in matrix gel group (r=0.92, P<0.05). On PID 14 and 21, the expressions of VEGF (with t values of 6.14 and 6.75, respectively, P<0.05) and EGF (with t values of 8.17 and 5.85, respectively, P<0.05) in wound tissue in matrix gel group were significantly higher than those in PBS group. Compared with the previous time point within the group, the expression of VEGF of wound at each time point after injury in the two groups was increased significantly (P<0.05), and the expression of EGF was decreased significantly (P<0.05). Conclusions: Adipose stem cell matrix gel may significantly promote the wound healing of full-thickness skin defects in rabbit ears by promoting collagen deposition and expressions of VEGF and EGF in wound tissue, and may further inhibit the scar hyperplasia after wound healing by inhibiting collagen deposition and expressions of TGF-β1 and α-SMA in scar tissue.

Mechanism of Der f 1/IGF-1 nanoparticle promoting the production of regulatory T cell / 临床耳鼻咽喉头颈外科杂志

Objective:To prepare PLGA nanoparticles loaded with Der f 1/IGF-1（Der f 1/IGF-1 NPs） and investigate their role in promoting the formation of Treg cells. Methods:NPs coated with Der f 1/IGF-1 were prepared by double emulsion method and their physicochemical properties and cumulative release rate in vitro were analyzed. After pretreatment, BMDC was divided into Saline group, Blank NPs group, Der f 1/IGF-1 group and Der f 1/IGF-1 NPs group. Determination of the expression of IL-10 and TGF-β in BMDC by ELISA. The number of Treg cells was detected by flow cytometry. Results:The results showed that Der f 1/IGF-1 NPs were spherical structures, with good dispersion, particle size less than 200 nm, negative charge and stable slow-release effect of Zeta potential. After BMDC pretreatment, the expression levels of TGF-β and IL-10 in BMDC cells in the Der f 1/IGF-1 NPs group were significantly increased compared with the Blank NPs group, and the difference was statistically significant（P<0.001）. After co-culture with CD4+ T cells, the proportion of Treg cells produced in the Der f 1/IGF-1 NPs group was significantly increased, and the difference was statistically significant（P<0.001）. Conclusion:Der f 1/IGF-1 NPs can induce Treg cell generation in vitro. This study provides a new and more effective method for the reconstruction of immune tolerance dysfunction.

Neotuberostemonine and tuberostemonine ameliorate pulmonary fibrosis through suppressing TGF-β and SDF-1 secreted by macrophages and fibroblasts via the PI3K-dependent AKT and ERK pathways / 中国天然药物

Activated fibroblasts and M2-polarized macrophages may contribute to the progression of pulmonary fibrosis by forming a positive feedback loop. This study was aimed to investigate whether fibroblasts and macrophages form this loop by secreting SDF-1 and TGF-β and the impacts of neotuberostemonine (NTS) and tuberostemonine (TS). Mice were intratracheally injected with 3 U·kg-1 bleomycin and orally administered with 30 mg·kg-1 NTS or TS. Primary pulmonary fibroblasts (PFBs) and MH-S cells (alveolar macrophages) were used in vitro. The animal experiments showed that NTS and TS improved fibrosis related indicators, inhibited fibroblast activation and macrophage M2 polarization, and reduced the levels of TGF-β and SDF-1 in alveolar lavage fluid. Cell experiments showed that TGF-β1 may activated fibroblasts into myofibroblasts secreting SDF-1 by activating the PI3K/AKT/HIF-1α and PI3K/PAK/RAF/ERK/HIF-1α pathways. It was also found for the first time that SDF-1 was able to directly polarize macrophages into M2 phenotype secreting TGF-β through the same pathways as mentioned above. Moreover, the results of the cell coculture confirmed that fibroblasts and macrophages actually developed a feedback loop to promote fibrosis, and the secretion of TGF-β and SDF-1 was crucial for maintaining this loop. NTS and TS may disturb this loop through inhibiting both the PI3K/AKT/HIF-1α and PI3K/PAK/RAF/ERK/HIF-1α pathways to improve pulmonary fibrosis. NTS and TS are stereoisomeric alkaloids with pyrrole[1,2-a]azapine skeleton, and their effect on improving pulmonary fibrosis may be largely attributed to their parent nucleus. Moreover, this study found that inhibition of both the AKT and ERK pathways is essential for maximizing the improvement of pulmonary fibrosis.

Amygdalin Ameliorates Liver Fibrosis through Inhibiting Activation of TGF-β/Smad Signaling / 中国结合医学杂志

OBJECTIVE@#To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro.@*METHODS@#Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl4)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor β (TGF-β)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed.@*RESULTS@#High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFβ R1, TGFβ R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01).@*CONCLUSIONS@#Amygdalin can dramatically alleviate liver fibrosis induced by CCl4 in mice and inhibit TGF-β/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.

Treg Cells, FoxP3 and TGF-β Expression and Significance in Chronic Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression and significance of regulatory T cells (Tregs), FoxP3 and transforming growth factor-β (TGF-β) in different phase of chronic myeloid leukemia (CML).@*METHODS@#Peripheral blood of 73 CML patients in Department of Hematology, Heze Municipal Hospital from March 2018 to March 2021 were collected. According to patient's period in CML, they were divided into ND CML group (newly diagnosed), CP CML group (chronic period), and BP CML group (blast phase). The percentage of Tregs, expression level of FoxP3 mRNA and TGF-β were detected by flow cytometry, RT-qPCR, and ELISA, respecitively. The roles of above indices in clinical pathogenesis of patients with CML were analyzed.@*RESULTS@#The proportion of Treg in the ND CML group was slightly higher than the CP CML group, but the difference was not statistically significant (P =0.695), while the BP CML group was significantly higher than the other two groups (P =0.008, P <0.001). The expression levels of FoxP3 mRNA in ND CML group, CP CML group and BP CML group were 11.61±2.21, 6.46±1.35 and 8.54±2.13, respectively. Significant difference in FoxP3 mRNA levels was observed among patients in different phases of CML (F =55.199, P <0.001). The expression levels of FoxP3 mRNA both in ND CML group and BP CML group were significantly higher than that in CP CML group (P <0.001), and the ND CML group was the highest (P <0.001). However, the expression levels of TGF-β in different phases of CML showed no statistical differences (H =0.634, P =0.728).@*CONCLUSION@#The abnormal distribution of Treg subset in different phases of CML and the significant increase of the expression level of FoxP3 mRNA in the new onset and blast phase of CML suggest that Tregs may promote the occurrence and progression of CML through immune regulation.

Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression / 细胞与分子免疫学杂志

Objective To investigate the effect of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) on the proliferation, migration and tumor immune microenvironment of colorectal cancer cells and its possible molecular mechanism. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of IGF2BP2 and MYC in colorectal cancer and adjacent tissues. The expression of IGF2BP2 in HCT-116 and SW480 human colorectal cancer cells was silenced by RNA interference (RNAi), and the silencing effect was detected by quantitative real-time PCR. After knocking down IGF2BP2, colony formation assay, CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were employed to detect cell colony formation and proliferation ability. TranswellTM assay was used to detect cell migration ability. Quantitative real-time PCR was used to detect the mRNA expression of IGF2BP2, MYC, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10). The protein expression of IGF2BP2 and MYC was detected by western blot. The binding ability of IGF2BP2 and MYC in HCT-116 cells was detected by quantitative real-time PCR after RNA immunoprecipitation. Results The results of TCGA database showed that the expression of IGF2BP2 and MYC in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the survival time of colorectal cancer patients with high expression of IGF2BP2 was shorter. After silencing IGF2BP2, the viability, proliferation and migration of HCT-116 and SW480 cells were decreased. The mRNA expression of MYC, TGF-β and IL-10 in IGF2BP2 knockdown group was significantly decreased, while the expression of TNF-α mRNA was increased. The expression of MYC protein and the stability of MYC mRNA were significantly decreased. RIP-qPCR results showed that IGF2BP2 could bind to MYC mRNA. Conclusion Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression.

Role of TGF-β/Smad signaling pathway in diabetic kidney disease and research progress of traditional Chinese medicine intervention / 中国中药杂志

Diabetic kidney disease is an important microvascular complication of diabetes and the leading cause of end-stage renal disease. Its pathological characteristics mainly include epithelial mesenchymal transition(EMT) in glomerulus, podocyte apoptosis and autophagy, and damage of glomerular filtration barrier. Transforming growth factor-β(TGF-β)/Smad signaling pathway is specifically regulated by a variety of mechanisms, and is a classic pathway involved in physiological activities such as apoptosis, proliferation and differentiation. At present, many studies have found that TGF-β/Smad signaling pathway plays a key role in the pathogenesis of diabetic kidney disease. Traditional Chinese medicine has significant advantages in the treatment of diabetic kidney disease for its multi-component, multi-target and multi-pathway characteristics, and some traditional Chinese medicine extracts, traditional Chinese medicines and traditional Chinese medicine compound prescription improve the renal injury of diabetic kidney disease by regulating TGF-β/Smad signaling pathway. This study clarified the mechanism of TGF-β/Smad signaling pathway in diabetic kidney disease by expounding the relationship between the key targets of the pathway and diabetic kidney disease, and summarized the research progress of traditional Chinese medicine in the treatment of diabetic kidney disease by interfering with TGF-β/Smad signaling pathway in recent years, to provide reference for drug research and clinical treatment of diabetic kidney disease in the future.

Ursolic acid increases strength in mdx mice model and may decrease fibrosis deposition by tgf-ß downregulation / El ácido ursólico aumenta la resistencia en el modelo de ratones mdx y podría disminuir la deposición de fibrosis mediante la regulación decreciente de tgf-ß

SUMMARY: Dystrophin disfunction results in sarcolemma destabilization, leading muscle cell damage by continuous degeneration cycles and limited regeneration. In muscle dystrophy, caused by dystrophin dysfunction, inflammation, necrosis and fibrosis are pathophysiological muscle function loss characteristics. As a genetic disease, this muscle dystrophy has no cure, however, advances in drug therapy using glucocorticoids can decrease the disease progression. Subsequently, alternative therapies were studied, such as ursolic acid (UA), that inhibits muscle atrophy and increases muscle mass and strength. Herein, we used 10 mg/kg daily supplementation in mdx mice for 4 weeks to evaluate serum creatine phosphokinase (CPK), muscle strength (Kondziela test), muscular organization (histology) and expression of fibrosis related genes (TGF-ß, TNF-α, mstn and ostn). UA supplementation increased muscle morphological organization, motor strength and decreased muscular TGF-ß expression. Altogether, the gene expression profile, histological organization and strength could suggest that UA treatment did not stop the fibrogenesis but decreased its progress.

RESUMEN: La disfunción de la distrofina resulta en la desestabilización del sarcolema, llevando al daño de las células musculares por ciclos continuos de degeneración y regeneración limitada. En la distrofia muscular, debido a la disfunción de la distrofina, la inflamación, la necrosis y la fibrosis, son características fisiopatológicas de la pérdida de la función muscular. Como enfermedad genetica no es possible remediar esta distrofia muscular, sin embargo, los avances en la terapia de medicamentos con glucocorticoides pueden disminuir la progresión de la enfermedad. Se estudiaron terapias alternativas, como el ácido ursólico (UA), que inhibe la atrofia muscular y aumenta la masa y la fuerza muscular. En este estudio, utilizamos una suplementación diaria de 10 mg / kg en ratones mdx durante 4 semanas para evaluar la creatina fosfoquinasa (CPK) sérica, la fuerza muscular (prueba de Kondziela), la organización muscular (histología) y la expresión de genes relacionados con la fibrosis (TGF-ß, TNF- α, mstn y ostn). La suplementación con AU aumentó la organización morfológica muscular, la fuerza motora y la disminución de la expresión muscular de TGF-ß. El perfil de expresión génica, la organización histológica y la fuerza simultáneamente podrían sugerir que el tratamiento con AU no detuvo la fibrogénesis sino que disminuyó su progreso.

Effects of laser photobiomodulation on tgf-ß and vegf expression in burn wound: systematic review and meta-analysis in the animal model / Influencia de la fotobiomodulación por láser en la expresión de tgf-ß y vegf en heridas por quemadura: revisión sistemática y meta-análisis en modelo animal

SUMMARY: Laser photobiomodulation (laser PBM) is known to be able to accelerate burn wound healing in the animal model; however little evidence exists on the action of laser PBM on the expression of important proteins in wound healing in the animal model, such as VEGF and TGF-ß1. The aim of this study was to carry out a systematic review in order to analyse the effect of laser PBM on VEGF and TGF-ß expression during burn wound repair in the animal model. A systematic review was carried out of the EMBASE, PubMed/ MEDLINE and LILACS databases. The studies included were preclinical studies that analysed the action of laser PBM on the expression of VEGF and TGF-ß (1, 2, 3) during burn wound repair in the animal model. The SYRCLE risk of bias tool was used. Random effect models were used to estimate the combined effect. Increased VEGF expression was observed with the use of laser PBM at 4.93 J/cm2 per point in the first two weeks after induction of the burn wound, with greater size of effect in the second week (SDM = 5.72; 95% CI: 3.14 to 8.31, I2 = 0 %; very low certainty of evidence). We also observed that the effect of laser PBM on TGF-ß1 expression was greater than in the control in the first week (SDM = -0.45; 95% CI: -1.91 to 1.02, I2 = 51 %; very low certainty of evidence), but diminished in the third week after induction of the lesion (SDM = -2.50; 95% CI: 3.98 to -1.01, I2 = 0 %; very low certainty of evidence). Laser PBM has an effect on TGF-ß1 and VEGF expression, promoting burn wound repair in the animal model.

RESUMEN: Es sabido que la fotobiomodulación por láser (FBM láser) puede acelerar el proceso de curación de heridas por quemadura en modelo animal, sin embargo aún se carece de mayor evidencia sobre la acción de la FBM láser en la expresión de proteínas importantes en el proceso de curación de heridas en modelo animal, como VEGF y TGF-ß1. Así, el objetivo de este estudio fue realizar una revisión sistemática a fin de analizar el efecto de la FBM láser sobre la expresión de VEGF, TGF-ß durante el proceso de reparación de heridas por quemadura en modelo animal. Se realizó una búsqueda sistemática en las bases de datos EMBASE, PubMed/MEDLINE y LILACS. Se incluyeron estudios preclínicos que analizaron la acción de la FBM láser en la expresión de VEGF, TGF-ß (1, 2, 3) durante el proceso de reparación de heridas por quemadura en modelo animal. Se utilizó la herramienta de riesgo de sesgo SYRCLE. Se utilizaron modelos de efectos aleatorios para estimar el efecto combinado. Observamos aumento de la expresión de VEGF con el uso de FBM láser 4.93 J/cm2 por punto, en las dos primeras semanas tras inducción de la herida por quemadura, con mayor tamaño de efecto en la segunda semana (SDM = 5,72; IC del 95%: 3,14 a 8,31, I2 = 0 %; certeza de la evidencia muy baja). También se observó el efecto de la FBM láser en la expresión del TGF- ß1 que fue mayor que el control en la primera semana (SDM = - 0,45; IC del 95%: -1,91 a 1,02, I2 = 51 %; certeza de la evidencia muy baja), disminuyendo en la tercera semana tras inducción de la lesión (SDM = -2,50; IC del 95%: -3,98 a -1,01; I2 = 0 %; certeza de la evidencia baja). La TFB por láser ejerce influencia en la expresión de TGF-ß1 y VEGF favoreciendo el proceso de reparación de heridas por quemadura en modelo animal.

O papel de proteínas das vias WNT/ß-Catenina, TGF-ß/BMP4 e SHH na odontogênese e odontomas / The role of proteins from the WNT/ß-Catenin, TGF-ß/BMP4 and SHH pathways in odontogenesis and odontomas

O desenvolvimento do dente depende de uma série de interações sinalizadoras recíprocas entre o epitélio oral (EO) e o ectomesênquima derivado da crista neural, a via WNT com o TGF-ß e BMP4 tem sido implicada na tumorigênese. A via de sinalização tipo Wingless (Wnt) / ß-catenina é essencial para a ativação precoce da odontogênese e no desenvolvimento de tumores odontogênicos. O TGF-ß e as BMPs tem sido associadas aos processos de dentinogênese reacionária e reparadora. A sinalização de Shh pode regular a proliferação celular no ectomesênquima dentário, controlando assim a morfogênese dentária. O objetivo da pesquisa foi investigar a atuação de algumas proteínas das vias na odontogênese e na formação de odontomas e tumores odontogênicos mistos benignos, para isto, foi desenvolvido um estudo seccional restrospectivo e imuno-histoquímico contendo 23 odontomas compostos, 21 odontomas complexos, 17 germes dentários, 05 fibro-odontomas ameloblásticos e 01 fibroma ameloblástico. Os resultados encontrados demonstraram maiores imunoexpressões da via WNT/ß-catenina no epitélio dos germes dentários (p<0,001) e no fibroma ameloblástico, enquanto que, esteve no ectomesênquima dos odontomas (p<0,001) e fibro-odontomas ameloblásticos. A via WNT/ßcatenina correlacionou-se moderadamente e significativamente com a CK14 no epitélio (p = 0,007) dos odontomas. A BMP4 foi imunoexpressa, especialmente, no ectomesênquima dos odontomas complexos (mediana = 33,7; p<0,001). A via Shh foi mais imunoexpressa no epitélio dos germes dentários (p<0,001) e no ectomesênquima dos odontomas complexos (p=0,029). De forma similar, o TGFß apresentou maior imunoexpressão no epitélio dos germes dentários (p<0,001) e no ectomesênquima dos odontomas complexos (p = 0,002). O dente em desenvolvimento exibiu maiores concentrações para estas proteínas no epitélio odontogênico nas fases de botão e capuz e a expressão diferencial ocorreu, principalmente, no ectomesênquima dos tumores, o que indica que esse componente é de fato mais proliferativo (AU).

Tooth development depends on a series of reciprocal signaling interactions between oral epithelium (EO) and neural crest-derived ectomesenchyme, the WNT pathway with TGF-ß and BMP4 has been implicated in tumorigenesis. The Wingless (Wnt)/ß-catenin signaling pathway is essential for the early activation of odontogenesis and the development of odontogenic tumors. TGF-ß and BMPs have been associated with reactionary and reparative dentinogenesis processes. Shh signaling can regulate cell proliferation in dental ectomesenchyme, thus controlling dental morphogenesis. The objective of the research was to investigate the role of some proteins in the pathways in odontogenesis and in the formation of odontomas and benign mixed odontogenic tumors. tooth germs, 05 ameloblastic fibro-odontomas and 01 ameloblastic fibroma. The results found showed higher immunoexpressions of the WNT/ß-catenin pathway in the epithelium of tooth germs (p<0.001) and in ameloblastic fibroma, while it was in the ectomesenchyme of odontomas (p<0.001) and ameloblastic fibroodontomas. The WNT/ß-catenin pathway correlated moderately and significantly with CK14 in the epithelium (p = 0.007) of odontomas. BMP4 was immunoexpressed, especially in the ectomesenchyme of complex odontomas (median = 33.7; p<0.001). The Shh pathway was more immunoexpressed in the epithelium of tooth germs (p<0.001) and in the ectomesenchyme of complex odontomas (p=0.029). Similarly, TGF-ß showed higher immunoexpression in the epithelium of tooth germs (p<0.001) and in the ectomesenchyme of complex odontomas (p = 0.002). The developing tooth exhibited higher concentrations of these proteins in the odontogenic epithelium in the bud and cap phases and the differential expression occurred mainly in the ectomesenchyme of the tumors, which indicates that this component is in fact more proliferative (AU).

Intermittent compressive force induces cell cycling and reduces apoptosis in embryoid bodies of mouse induced pluripotent stem cells / 国际口腔科学杂志·英文版

In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.

Efficacy of Qingfei oral liquid for idiopathic pulmonary fibrosis in rats and related network pharmacology study / 浙江大学学报·医学版

To investigate the therapeutic effect and mechanism of Qingfei oral liquid in idiopathic pulmonary fibrosis. Seventy-two male SD rats were divided into control group, model group, pirofenidone group and Qingfei group with 18 animals in each group. The idiopathic pulmonary fibrosis was induced in last three groups by intratracheal injection of bleomycin; pirofenidone group was given oral administration of pirofenidone b.i.d for 21 d, and Qingfei group was given Qingfei oral liquid 3.6 mL/kg q.d for Lung tissues were obtained for HE staining, Masson staining and transforming growth factor (TGF)-β immunohistochemical staining. Superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) were detected in tissue homogenates. The BATMAN-TCM database was used to retrieve the chemical components and their corresponding targets of Qingfei oral solution by network pharmacology method, and then the component-target-disease network diagram was constructed. Finally, the pathway enrichment analysis was carried out to explore the molecular mechanism of Qingfei oral liquid against idiopathic fibrosis. Histopathology results showed that Qingfei oral liquid had a similar relieving effect on pulmonary fibrosis as the positive drug pirfenidone; TGF-β secretion had a significant reduction in lung tissues of Qingfei group; and Qingfei oral liquid had better regulatory effect on SOD, MDA and GSH than pirfenidone. The results of component-target-disease network and pathway enrichment analysis showed that the related molecular pathways were concentrated in inflammation, extracellular matrix and cytokines. Qingfei oral liquid has a good therapeutic effect on idiopathic pulmonary fibrosis in rats via regulation of inflammation, extracellular matrix and cytokines.

Vasohibin-2 promotes proliferation and metastasis of cervical cancer cells by regulating epithelial-mesenchymal transition / 南方医科大学学报

OBJECTIVE@#To explore the role of vasohibin-2 (VASH2) in regulation of proliferation and metastasis of cervical cancer cells.@*METHODS@#We analyzed the differentially expressed genes between cervical cancer cells with flotillin-1 overexpression and knockdown by RNA-seq combined with analysis of public databases. The expression levels of VASH2 were examined in normal cervical epithelial cells (HcerEpic), cervical cancer cell lines (HeLa, C-33A, Ca ski, SiHa and MS751) and fresh cervical cancer tissues with different lymph node metastasis status. We further tested the effects of lentivirus-mediated overexpression and interference of VASH2 on proliferation, migration, invasion and lymphatic vessel formation of the cervical cancer cells and detected the expression levels of key epithelial-mesenchymal transition (EMT) markers and TGF-β mRNA.@*RESULTS@#RNA-seq and analysis of public databases showed that VASH2 expression was significantly upregulated in cervical cancer cells exogenously overexpressing flotillin-1 (P < 0.05) and downregulated in cells with flotillin-1 knockdown (P < 0.05), and was significantly higher in cervical cancer tissues with lymph node metastasis than in those without lymph node metastasis (P < 0.01). In cervical cancer cell lines Ca Ski, SiHa, and MS751 and cervical cancer tissue specimens with lymph node metastasis, VASH2 expression was also significantly upregulated as compared with HcerEpic cells and cervical cancer tissues without lymph node metastasis (P < 0.05). Exogenous overexpression of VASH2 significantly promoted proliferation, migration, invasion and lymphatic vessel formation of cervical cancer cells, whereas these abilities were significantly inhibited in cells with VASH2 knockdown (P < 0.05). The cervical cancer cells overexpressing VASH2 showed significant down- regulation of e-cadherin and up- regulation of N-cadherin, Vimentin and VEGF-C, while the reverse changes were detected in cells with VASH2 knockdown (P < 0.05). TGF-β mRNA expression was significantly up-regulated in cervical cancer cells overexpressing VASH2 and down-regulated in cells with VASH2 knockdown (P < 0.001).@*CONCLUSION@#Flotillin-1 may participate in TGF-β signaling pathway-mediated EMT through its down-stream target gene VASH2 to promote the proliferation, migration, invasion and lymphatic vessel formation of cervical cancer cells in vitro.

Protective effect of excretory-secretory proteins from Trichinella spiralis muscle larvae against myocardial injury in septic mice / 南方医科大学学报

OBJECTIVE@#To evaluate the protective effect of excretory-secretory proteins from Trichinella spiralis muscle larvae (Ts-MES) on sepsis-induced myocardial injury in mice.@*METHODS@#Eighty male BALB/C mice were randomized equally into sham-operated group, myocardial injury group, Ts-MES treatment group and dexamethasone treatment group. In the latter 3 groups, sepsis-induced myocardial injury models were established by cecal ligation and perforation; the sham operation was performed by exposure of the cecum without ligation or perforation. Forty minutes after the operation, the mice were given intraperitoneal injections 150 μL PBS, 20 μg TS-MES or 0.3 mg/kg dexamethasone as indicated. At 12 h after the operation, 6 mice were randomly selected from each group for echocardiography, and 8 mice were used for observing the survival rate within 72 h. The remaining 6 mice were examined for myocardial pathologies with HE staining and serum levels of NTPro-BNP and cTnI with ELISA; the expressions of TNF-α, IL-6, IL-10 and TGF-β in the serum and myocardial tissue were detected using ELISA and qRT-PCR.@*RESULTS@#Compared with the sham-operated mice, the septic mice showed significantly decreased cardiac function indexes (LVEF, LVFS, and E/A) with lowered survival rate within 72 h (P < 0.001) and significantly higher myocardial injury scores and serum levels of NTPro-BNP and cTnI (P < 0.01). Treatment with TS-MES significantly improved the cardiac function and 72-h survival rate (P < 0.05) and lowered the myocardial injury scores and serum levels of NTPro-BNP and cTnI (P < 0.05) in the septic mice. Compared with the sham-operated mice, the septic mice had obviously increased TNF-α and IL-6 levels in the serum and myocardial tissue (P < 0.001), which were significantly lowered by treatment with TS-MES (P < 0.05). TS-MES and dexamethasone both increased the levels of IL-10 and TGF-β in the septic mice, but the changes were significant only in TS-MES-treated mice (P < 0.05).@*CONCLUSION@#Ts-MES are capable of protecting against myocardial injury in septic mice by reducing the production of pro-inflammatory cytokines and enhancing the levels of regulatory cytokines.

PTHrP promotes subchondral bone formation in TMJ-OA / 国际口腔科学杂志·英文版

PTH-related peptide (PTHrP) improves the bone marrow micro-environment to activate the bone-remodelling, but the coordinated regulation of PTHrP and transforming growth factor-β (TGFβ) signalling in TMJ-OA remains incompletely understood. We used disordered occlusion to establish model animals that recapitulate the ordinary clinical aetiology of TMJ-OA. Immunohistochemical and histological analyses revealed condylar fibrocartilage degeneration in model animals following disordered occlusion. TMJ-OA model animals administered intermittent PTHrP (iPTH) exhibited significantly decreased condylar cartilage degeneration. Micro-CT, histomorphometry, and Western Blot analyses disclosed that iPTH promoted subchondral bone formation in the TMJ-OA model animals. In addition, iPTH increased the number of osterix (OSX)-positive cells and osteocalcin (OCN)-positive cells in the subchondral bone marrow cavity. However, the number of osteoclasts was also increased by iPTH, indicating that subchondral bone volume increase was mainly due to the iPTH-mediated increase in the bone-formation ability of condylar subchondral bone. In vitro, PTHrP treatment increased condylar subchondral bone marrow-derived mesenchymal stem cell (SMSC) osteoblastic differentiation potential and upregulated the gene and protein expression of key regulators of osteogenesis. Furthermore, we found that PTHrP-PTH1R signalling inhibits TGFβ signalling during osteoblastic differentiation. Collectively, these data suggested that iPTH improves OA lesions by enhancing osteoblastic differentiation in subchondral bone and suppressing aberrant active TGFβ signalling. These findings indicated that PTHrP, which targets the TGFβ signalling pathway, may be an effective biological reagent to prevent and treat TMJ-OA in the clinic.

The role and molecular mechanism of miR-494 in disease development and progression / 生理学报

MicroRNA-494 (miR-494) is a small non-coding RNA located in chromosome 14q32.31 and regulates post-transcriptional gene expression by promoting the degradation of its target mRNAs via binding to the 3' untranslated regions (3'UTR). It has been reported that miR-494 plays an important role in the occurrence, development and prognosis of various diseases. Several signaling pathways modulated by miR-494 including the PTEN/PI3K/AKT, nuclear factor κ-B (NF-κB), mitogen-activated protein kinase (MAPK), transforming growth factor-β (TGF-β)/SMAD, and Wnt/β-catenin are associated with physiological regulation and pathological process in many diseases. The stably expression of miR-494 in the blood stream suggests its potential as a biological marker for disease diagnosis, treatment, and prognosis. Based on recent research, we summarize the role and molecular mechanism of miR-494 in disease development and progression. We also discuss its potential as a marker for clinical diagnosis and prognosis of various diseases.