Strictosamide promotes wound healing through activation of the PI3K/AKT pathway.

Nauclea officinalis, as a Chinese medicine in Hainan province, had the effect of treating lower limb ulcers, burn infections. In this paper, we studied the effect of Strictosamide (STR), the main bioactive compound in Nauclea officinals, on wound healing and explored its internal mechanism. Firstly, the wound healing potential of STR was evaluated in a rat model, demonstrating its ability to expedite wound healing, mitigate inflammatory infiltration, and enhance collagen deposition. Additionally, immunofluorescence analysis revealed that STR up-regulated the expression of CD31 and PCNA. Subsequently, target prediction, protein-protein interaction (PPI), gene ontology (GO), and pathway enrichment analyses were used to obtain potential targets, specific biological processes, and molecular mechanisms of STR for the potential treatment of wound healing. Furthermore, molecular docking was conducted to predict the binding affinity between STR and its associated targets. Additionally, in vivo and in vitro experiments confirmed that STR could increase the expression of P-PI3K, P-AKT and P-mTOR by activating the PI3K/AKT signaling pathway. In summary, this study provided a new explanation for the mechanism by which STR promotes wound healing through network pharmacology, suggesting that STR may be a new candidate for treating wound.

Strictosamide alleviates acute lung injury via regulating T helper 17 cells, regulatory T cells, and gut microbiota.

BACKGROUND: Nauclea officinalis (Pierre ex Pit.) Merr. & Chun (Rubiaceae) is widely used to treat respiratory diseases in China. Strictosamide is its main active component and has significant anti-inflammatory activity. However, the effects and molecular mechanisms of strictosamide in the treatment of acute lung injury (ALI) remain largely unknown. PURPOSE: This study aimed to examine the regulatory effects of strictosamide on T helper 17 cells (Th17 cells)/Regulatory T cells (Treg cells) and gut microbiota in ALI-affected mice. MATERIALS AND METHODS: The ALI model was induced using lipopolysaccharide (LPS) intraperitoneal injection. Hematoxylin-eosin (H&E) staining, the number of inflammatory cells in broncho-alveolar lavage fluid (BALF), the Wet/Dry (W/D) ratio, and myeloperoxidase (MPO) activity were utilized as evaluation indices for the therapeutic efficacy of strictosamide on ALI. Flow cytometry (FCM), enzyme-linked immune sorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blotting were used to determine the regulation of strictosamide on the Th17/Treg cells and the STAT3/STAT5 signaling pathway. The analysis of gut microbiota was conducted using 16S rDNA sequencing. The verification of the relationship between the gut microbiome and immune function was conducted using Spearman analysis. RESULTS: Strictosamide attenuated inflammation on ALI induced by LPS, which reduced the levels of Th17-related factors interleukin (IL)-6 and IL-17 and increased Treg-related factors IL-10 and transforming growth factor (TGF)-ß. In the spleens and whole blood, strictosamide reduced the proportion of Th17 cells and increased the proportion of Treg cells. Furthermore, strictosamide increased Forkhead/winged helix transcription factor 3 (Foxp3) and p-STAT5 protein expression while inhibiting Retinoid-related orphan nuclear receptors-γt (RORγt) and p-STAT3 expression. Moreover, strictosamide reshaped the diversity and structure of the gut microbiota, and influence the associations between immune parameters and gut microbiota in ALI mice. CONCLUSIONS: In summary, the results of the current investigation showed that strictosamide has a therapeutic impact on LPS-induced ALI. The mechanism of action of this effect may be associated with the modulation of Th17 and Treg cells differentiation via the SATA signaling pathway, as well as the impact of the gut microbiota.

Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair.

OBJECTIVE: To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. METHODS: Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 ß in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. RESULTS: DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 ß (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). CONCLUSIONS: DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.

Sympathetic Neurostress Drives Osteoblastic Exosomal MiR-21 Transfer to Disrupt Bone Homeostasis and Promote Osteopenia.

Innervation and extracellular vesicle secretion co-exist in the local tissue microenvironment for message transfer, but whether they are interconnected to regulate organ homeostasis remains unknown. Sympatho-adrenergic activation is implicated in stress-induced depression and leads to bone loss, but the mechanisms and therapeutics are incompletely elucidated. Here, it is revealed that sympathetic neurostress through the ß1/2 -adrenergic receptor (ß1/2-AR) signaling triggers the transcription response of a microRNA, miR-21, in osteoblasts, which is transferred to osteoclast progenitors via exosomes for dictating osteoclastogenesis. After confirming that miR-21 deficiency retards the ß1/2-AR agonist isoproterenol (ISO)-induced osteopenia, it is shown that the pharmacological inhibition of exosome release by two clinically-relevant drugs, dimethyl amiloride and omeprazole, suppresses osteoblastic miR-21 transfer and ameliorates bone loss under both ISO and chronic variable stress (CVS)-induced depression conditions. A targeted delivery approach to specifically silence osteoblastic miR-21 is further applied, which is effective in rescuing the bone remodeling balance and ameliorating ISO- and CVS-induced osteopenias. These results decipher a previously unrecognized paradigm that neural cues drive exosomal microRNA communication to regulate organ homeostasis and help to establish feasible strategies to counteract bone loss under psychological stresses.

Feasibility analysis of CT-guided thermal ablation of multiple pulmonary nodules combined with intraoperative biopsy.

Purpose: To analyze the safety and feasibility of computed tomography (CT)-guided thermal ablation of multiple pulmonary nodules combined with intraoperative biopsy. Methods: The data of 431 patients with 540 lung nodules undergoing CT-guided biopsy or ablation were retrospectively analyzed. Biopsy-only group (A): 107 patients (107 lesions) received CT-guided percutaneous lung biopsy only; Ablation-only group (B): 117 cases (117 lesions) only received CT-guided thermal ablation; Single focal ablation combined with biopsy group (C): 103 patients (103 lesions) received CT-guided thermal ablation combined with intraoperative immediate biopsy; Multifocal ablation combined with biopsy group (D): 104 patients (213 lesions) received CT-guided thermal ablation combined with intraoperative biopsy. The success rate of this technique was calculated, the complications were recorded, and the positive rate of pathological diagnosis of the specimens was evaluated (the tissue specimens could be confirmed as positive by pathological diagnosis). Results: All 431 patients with pulmonary nodules successfully completed the operation, and the technical success rate was 100% (431/431). In group A, hemoptysis occurred in seven cases after operation, while no hemoptysis was observed in the other groups. Pneumothorax occurred in 8 cases in group A, 14 cases in group B, 11 cases in group C, and 13 cases in group D. Hydrothorax occurred in 4 cases in group A, 7 cases in group B, 5 cases in group C and 9 cases in group D, and there were no significant differences between the groups. The positive rate of pathological diagnosis was 84.1% (90/107) in group A, 81.5% (84/103) in group C, and 82.6% (176/213) in group D, and there was no significant difference among the groups (P > 0.05). A total of 15 cases in group C and 23 cases in group D underwent gene testing and analysis, and the biopsy tissue samples all met quality control standards. Conclusion: CT-guided thermal ablation of multiple pulmonary nodules combined with intraoperative biopsy does not prolong the length of hospital stay or increase the risk of postoperative complications. It can meet the requirements of clinical, pathological and genetic testing, and is safe and reliable.

Endoplasmic Reticulum and Its Significance in Periodontal Disease.

The endoplasmic reticulum has emerged as a modulator that is essential for cellular homeostasis and human health. It is an extensive membranous organelle that acts as a hub for the physiological and pathological processes. In recent years, it has become a topic of interest in studies on the relationship between endoplasmic reticulum homeostasis and system diseases. Periodontal disease is a prevalent chronic disease that affects tooth-supporting tissues, initiated by the interaction between pathogenic bacterial infection and immune defence and resulting in tooth loss. The endoplasmic reticulum participates in the responses to the fluctuating microenvironments in periodontal pathogenesis and regulates periodontal homeostasis. In this review, we present an overview of the significance of endoplasmic reticulum regulation as a multidimensional mediator in periodontal disease and highlight the potential strategies for periodontal regeneration.

Do Rural Residents in China Understand EQ-5D-5L as Intended? Evidence From a Qualitative Study.

BACKGROUND: The 5-level EQ-5D (EQ-5D-5L) has been increasingly used in China to measure the health status of the general population and patients. However, its content validity among rural residents in China has not been formally evaluated. This qualitative study aims to assess the content validity of EQ-5D-5L among rural Chinese. METHODS: Participants were recruited from four regions (North, South, East and West) across China. Eligible participants were those living in the rural area in last three years and making a living by agricultural operations. Semi-structured interviews were conducted. Interview transcripts were analysed to assess the comprehensibility, relevance, clarity and comprehensiveness. RESULTS: Sixty-two participants were included, comparable to the national figures regarding age, sex and education. For comprehensibility, participants could understand the 'mobility', 'self-care' and 'usual activities' domains well, but some reported confusions in 'pain/discomfort' (n = 42) and 'anxiety/depression' (n = 35). Some also reported difficulties in understanding anxiety (n = 6) and depression (n = 9), possibly due to the formal wording used. For relevance, all domains were reported as health-related and participants' responses were based on their own health. For clarity, all could distinguish the five levels, but suggestions on reducing response levels and alternative wording for 'slight' were raised. For comprehensiveness, two aspects (fatigue/energy and appetite) were raised beyond the EQ-5D-5L domains. The 'mobility' domain was selected as the most important and 'anxiety/depression' as the least important. CONCLUSION: Rural Chinese reported problems on the content validity of Chinese EQ-5D-5L. It might be sensible to consider some revisions to make it more understandable for rural residents.

Sensory nerve-deficient microenvironment impairs tooth homeostasis by inducing apoptosis of dental pulp stem cells.

OBJECTIVES: The aim of this study is to investigate the role of sensory nerve in tooth homeostasis and its effect on mesenchymal stromal/stem cells (MSCs) in dental pulp. MATERIALS AND METHODS: We established the rat denervated incisor models to identify the morphological and histological changes of tooth. The groups were as follows: IANx (inferior alveolar nerve section), SCGx (superior cervical ganglion removal), IANx + SCGx and Sham group. The biological behaviour of dental pulp stromal/stem cells (DPSCs) was evaluated. Finally, we applied activin B to DPSCs from sensory nerve-deficient microenvironment to analyse the changes of proliferation and apoptosis. RESULTS: Incisor of IANx and IANx + SCGx groups exhibited obvious disorganized tooth structure, while SCGx group only showed slight decrease of dentin thickness, implying sensory nerve, not sympathetic nerve, contributes to the tooth homeostasis. Moreover, we found sensory nerve injury led to disfunction of DPSCs via activin B/SMAD2/3 signalling in vitro. Supplementing activin B promoted proliferation and reduced apoptosis of DPSCs in sensory nerve-deficient microenvironment. CONCLUSIONS: This research first demonstrates that sensory nerve-deficient microenvironment impairs tooth haemostasis by inducing apoptosis of DPSCs via activin B/SMAD2/3 signalling. Our study provides the evidence for the crucial role of sensory nerve in tooth homeostasis.

L-type voltage-gated calcium channels in stem cells and tissue engineering.

L-type voltage-gated calcium ion channels (L-VGCCs) have been demonstrated to be the mediator of several significant intracellular activities in excitable cells, such as neurons, chromaffin cells and myocytes. Recently, an increasing number of studies have investigated the function of L-VGCCs in non-excitable cells, particularly stem cells. However, there appear to be no systematic reviews of the relationship between L-VGCCs and stem cells, and filling this gap is prescient considering the contribution of L-VGCCs to the proliferation and differentiation of several types of stem cells. This review will discuss the possible involvement of L-VGCCs in stem cells, mainly focusing on osteogenesis mediated by mesenchymal stem cells (MSCs) from different tissues and neurogenesis mediated by neural stem/progenitor cells (NSCs). Additionally, advanced applications that use these channels as the target for tissue engineering, which may offer the hope of tissue regeneration in the future, will also be explored.

Recipient Glycemic Micro-environments Govern Therapeutic Effects of Mesenchymal Stem Cell Infusion on Osteopenia.

Therapeutic effects of mesenchymal stem cell (MSC) infusion have been revealed in various human disorders, but impacts of diseased micro-environments are only beginning to be noticed. Donor diabetic hyperglycemia is reported to impair therapeutic efficacy of stem cells. However, whether recipient diabetic condition also affects MSC-mediated therapy is unknown. We and others have previously shown that MSC infusion could cure osteopenia, particularly in ovariectomized (OVX) mice. Here, we discovered impaired MSC therapeutic effects on osteopenia in recipient type 1 diabetes (T1D). Through intensive glycemic control by daily insulin treatments, therapeutic effects of MSCs on osteopenia were maintained. Interestingly, by only transiently restoration of recipient euglycemia using single insulin injection, MSC infusion could also rescue T1D-induced osteopenia. Conversely, under recipient hyperglycemia induced by glucose injection in OVX mice, MSC-mediated therapeutic effects on osteopenia were diminished. Mechanistically, recipient hyperglycemic micro-environments reduce anti-inflammatory capacity of MSCs in osteoporotic therapy through suppressing MSC interaction with T cells via the Adenosine monophosphate-activated protein kinase (AMPK) pathway. We further revealed in diabetic micro-environments, double infusion of MSCs ameliorated osteopenia by anti-inflammation, attributed to the first transplanted MSCs which normalized the recipient glucose homeostasis. Collectively, our findings uncover a previously unrecognized role of recipient glycemic conditions controlling MSC-mediated therapy, and unravel that fulfillment of potent therapeutic effects of MSCs requires tight control of recipient micro-environments.

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cells in vitro model.

Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.

Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro.

Myostatin is an important negative regulator of muscle growth and development. Natural mutations of the myostatin gene cause a double muscling phenotype in beef cattle, pigs and sheep. Therefore, it is feasible to produce a high growth domestic breed by generating a transgenic animal with a mutation, deletion or knockout of the myostatin gene. Our objective was to introduce a subtle mutation of G to A 281-bp upstream of the 3' untranslated region (3'UTR) end of the myostatin gene in Poll Dorset fetal myoblast cells in vitro. Fetal myoblast cells were isolated from fetuses at day 50 of gestation from Poll Dorset sheep and transfected with linear gene-targeting vector pMSTN-A using electroporation. We obtained seven gene-targeted cell colonies with homologous recombination, which were positive as confirmed by PCR, Southern blot. The Western blot analysis result demonstrated that the myostatin protein expression in positive colonies is lower than that of negative ones. These results strongly suggest that we successfully mutated the myostatin gene of Poll Dorset ovine fetal myoblast cells and the mutation can effectively downregulate the myostatin protein expression.

Multipotent differentiation of the EGFP gene transgenic stem cells derived from amniotic fluid of goat at terminal gestational age.

We have isolated stem cells from amniotic fluid of goat at terminal gestational age and transferred the EGFP (enhanced green fluorescent protein) gene into the stem cells previously. The aim of this study was to determine whether the transgenic stem cells have the capability of multipotent differentiation. The transgenic stem cells were induced to differentiate into neurogenic, adipogenic, osteogenic and endothelial cells in vitro. Markers associated with AFS (amniotic fluid-derived stem) cells and the differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that the transgenic AFS cells were capable of self-renewal, a defining property of stem cells. AFS cells were positive for the undifferentiated cell markers, Oct4, Nanog, Sox2 and Hes1, while following differentiation cells expressed markers for neurogenic cells such as astrocyte [GFAP (glial fibrillary acidic protein)] and NSE (neuron-specific enolase), adipogenic cells [LPL+ (lipoprotein lipase+)], osteogenic cells (osteocalcin+ and osteonectin+) and endothelium [CD34+ and eNOS+ (endothelial nitric oxide synthase)]. The results demonstrated that the EGFP gene transgenic AFS cells have the capability of multipotent differentiation, which means that the transgenic AFS cells may be useful in cell-transplantation studies in future.

Recombinant adenovirus-mediated human telomerase reverse transcriptase gene can stimulate cell proliferation and maintain primitive characteristics in bovine mammary gland epithelial cells.

The human telomerase reverse transcriptase (hTERT) gene has been used to stimulate the proliferation of most types of human cells. The present study was designed to evaluate the feasibility and efficiency of adenovirus-mediated hTERT in the proliferation of bovine mammary gland epithelial cells (bMGEs). A plasmid and an adenovirus vector that carried hTERT, namely pEGFP- hTERT and Ad- hTERT, were constructed and transfected into bMGEs, respectively. In order to select the best strategy for stimulating cell proliferation, the adenovirus- and plasmid-mediated hTERT were compared in terms of the positive cloning and transgenic efficiency. The results showed that only Ad- hTERT had high infection efficiency and produced a positive polyclone population (hTERT-bMGEs). The characteristics of the hTERT-bMGEs were investigated with further analysis by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, proliferation assays, and flow cytometry, which showed that hTERT facilitated strong cell proliferation. Real-time quantitative PCR showed a normal level of expression of beta-casein, the caspase-8 and c-myc proto-oncogene, and immunofluorescence demonstrated the properties of the epithelial cells. In conclusion, the adenovirus-mediated hTERT gene could not only extend the cell lifespan, but also maintained the primary characteristics of the cells. It may be possible to extend the use of a wide variety of non-human mammalian cells in this way. This study has provided additional insight into the mechanism of cell proliferation by demonstrating the lack of integration of the adenovirus-mediated hTERT gene into the mammalian genome.

Comparation of enhanced green fluorescent protein gene transfected and wild-type porcine neural stem cells.

The aim of this study was to transfect and express the enhanced green fluorescence protein (EGFP) gene into porcine neural stem cells (NSCs) to determine whether EGFP can be used as a marker to monitor NSCs. NSCs were isolated from embryonic day 30 fetal pig brain and transfected with EGFP gene using lipofection. Transfected and wild-type NSCs were induced to differentiate into cells of neuronal and myogenic lineages. Markers of passage three NSCs and their differentiated cells were tested by reverse transcription polymerase chain reaction. The results showed that EGFP could be expressed in NSCs and the differentiated cells. NSCs expressed Nestin, NogoA, DCX, Hes1, Oct4, CD-90 and Sox2. NSCs could differentiated into astrocyte (GFAP(+)), oligodendrocyte (GalC(+)), neuron (NF(+), NSE(+) and MAP2(+)) and myocyte (myf-6(+) and myoD(+)). We concluded that EGFP can be used as a marker in monitoring NSCs.

Development of cloned embryos from porcine neural stem cells and amniotic fluid-derived stem cells transfected with enhanced green fluorescence protein gene.

We assessed the developmental ability of embryos cloned from porcine neural stem (NS) cells, amniotic fluid-derived stem (AFS) cells, fetal fibroblast cells, adult fibroblast, and mammary gland epithelial cells. The five cell lines were transfected with enhanced green fluorescence protein gene respectively using lipofection. NS and AFS cells were induced to differentiate in vitro. Stem cells and their differentiated cells were harvested for analysis of the markers using RT-PCR. The five cell lines were used for nuclear transfer. The two-cell stage-cloned embryos derived from each cell line were transferred into the oviducts of surrogate mothers. The results showed that both NS and AFS cells expressed POU5F1, THY1 and SOX2, and they were both induced to differentiate into astrocyte (GFAP+), oligodendrocyte (GalC+), neuron (NF+, ENO2+, and MAP2+), adipocyte (LPL+ and PPARG-D+), osteoblast (osteonectin+ and osteocalcin+), myocyte (MYF6+ and MYOD+), and endothelium (PECAM1+, CD34+, CDH5+, and NOS3+) respectively. Seven cloned fetuses (28 days and 32 days) derived from stem cells were obtained. The in vitro developmental ability (morula-blastocyst rate was 28.26-30.07%) and in vivo developmental ability (pregnancy rate were 1.67-2.17%) of the embryos cloned from stem cells were higher (P<0.05) than that of the embryos cloned from somatic cells (morula-blastocyst rate was 16.27-19.28% and pregnancy rate was 0.00%), which suggests that the undifferentiated state of the donor cells increases cloning efficiency.

Immune response induced by spike protein from transmissible gastroenteritis coronavirus expressed in mouse mammary cells.

The present study is undertaken to investigate the immune response that was induced by the recombinant spike (S) protein from swine-transmissible gastroenteritis virus (TGEV) expressed in mouse mammary cells. A mammary-specific expression vector pEBS containing the full-length cDNA of S gene was constructed and expressed in the mouse mammary cells (EMT6). The recombinant S protein from culture supernatant of transgenic EMT6 was harvested and immunized BALB/c mice. The results demonstrated recombinant S protein was expressed at high levels in mammary cells by Western blotting and enzyme-linked immunosorbent assay (ELISA) detection. The antibody titer in BALB/c mice following immunization with recombinant S protein was detectable after the first immunization. Maximum titers of antibody (8.86+/-0.19 ng/ml of serum) were attained after the second immunization. In conclusion, the recombinant S protein expressed in mammary cells was able to elicit substantial immunological response against TGEV. This lays the basis for using mammary gland bioreactor generating edible vaccine.