Ursolic acid molecules dock MAPK1 to modulate gut microbiota diversity to reduce neuropathic pain.

To investigate the efficacy of Ursolic acid in alleviating neuropathic pain in rats with spinal nerve ligation (SNL), the SNL rat model was surgically induced. Different concentrations of Ursolic acid and manipulated target mitogen-activated protein kinase 1 (MAPK1) were administered to the SNL rats. Fecal samples were collected from each group of rats for 16S rDNA analysis to examine the impact of gut microbiota. Molecular docking experiments were conducted to assess the binding energy between Ursolic acid and MAPK1. In vivo studies were carried out to evaluate the expression of inflammatory factors and signaling pathways in spinal cord and colon tissues. Ursolic acid was found to have a beneficial effect on pain reduction in rats by increasing plantar withdrawal latency (PWL) and paw withdrawal threshold (PWT). Comparing the Ursolic acid group with the control group revealed notable differences in the distribution of Staphylococcus, Allobaculum, Clostridium, Blautia, Bifidobacterium, and Prevotella species. Network pharmacology analysis identified MAPK1 and intercellular adhesion molecule-1 (ICAM1) as common targets for Ursolic acid, SNL, and neuropathic pain. Binding sites between Ursolic acid and these targets were identified. Additionally, immunofluorescent staining showed a decrease in GFAP and IBA1 intensity in the spinal cord along with an increase in NeuN following Ursolic acid treatment. Overexpression of MAPK1 in SNL rats led to an increase in inflammatory factors and a decrease in PWL and PWT. Furthermore, MAPK1 counteracted the pain-relieving effects of Ursolic acid in SNL rats. Ursolic acid was found to alleviate neuropathic pain in SNL rats by targeting MAPK1 and influencing gut microbiota homeostasis.

Cross­talk between lymphangiogenesis and malignant melanoma cells: New opinions on tumour drainage and immunization (Review).

Malignant melanoma (MM) is a highly aggressive tumour that can easily metastasize through the lymphatic system at the early stages. Lymph node (LN) involvement and lymphatic vessel (LV) density (LVD) represent a harbinger of an adverse prognosis, indicating a strong link between the state of the lymphatic system and the advancement of MM. Permeable capillary lymphatic vessels are the optimal conduits for melanoma cell (MMC) invasion, and lymphatic endothelial cells (LECs) can also release a variety of chemokines that actively attract MMCs expressing chemokine ligands through a gradient orientation. Moreover, due to the lower oxidative stress environment in the lymph compared with the blood circulation, MMCs are more likely to survive and colonize. The number of LVs surrounding MM is associated with tumour-infiltrating lymphocytes (TILs), which is crucial for the effectiveness of immunotherapy. On the other hand, MMCs can release various endothelial growth factors such as VEGF-C/D-VEGFR3 to mediate LN education and promote lymphangiogenesis. Tumour-derived extracellular vesicles are also used to promote lymphangiogenesis and create a microenvironment that is more conducive to tumour progression. MM is surrounded by a large number of lymphocytes. However, both LECs and MMCs are highly plastic, playing multiple roles in evading immune surveillance. They achieve this by expressing inhibitory ligands or reducing antigen recognition. In recent years, tertiary lymphoid structures have been shown to be associated with response to anti-immune checkpoint therapy, which is often a positive prognostic feature in MM. The present review discusses the interaction between lymphangiogenesis and MM metastasis, and it was concluded that the relationship between LVD and TILs and patient prognosis is analogous to a dynamically tilted scale.

Relationship Between the Triglyceride-Glucose Index and Type 2 Diabetic Macroangiopathy: A Single-Center Retrospective Analysis.

Purpose: The research explores the relationship between the triglyceride-glucose index (TyG index) and the macroangiopathy risk in single-center hospitalized type 2 diabetes mellitus (T2DM) patients and develops a risk prediction nomogram model. Patients and Methods: A total of 858 patients with T2DM were studied retrospectively. Lasso regression was used to eliminate unimportant factors, and multivariate logistic regression analysis was used to investigate the association between the TyG index and macrovascular disease in T2DM. A nomogram model was constructed to predict macrovascular disease in T2DM and tested using the bootstrap technique, and the efficacy of the nomogram model was investigated using ROC curves. The multivariate Cox proportional hazards model estimated the association between the TyG index and all-cause mortality. Results: TyG index, high-density lipoprotein, red blood cell count, hypertension, history of taking ACEI/ARB drugs, and aortic calcification were closely related to macrovascular complications. In Cox proportional hazard model, the HRs of TyG index were 1.89 (95% confidence interval (CI) 1.29-2.76, p < 0.001) after adjusting for covariates. The risk of all-cause mortality in T2DM with macrovascular complications was significantly higher than in diabetic patients without vascular disease. In the ROC curve analysis, the cut-off value of the TyG index for macrovascular complications of T2DM was 9.31 (AUC: 0.702, 95% CI 0.67-0.74, p < 0.001). Conclusion: TyG index predicts future macrovascular disease in diabetic patients independently of known cardiovascular risk factors, suggesting that TyG index may be a useful marker for prognosis in diabetic patients.

Ursolic Acid Ameliorates Spinal Cord Injury in Mice by Regulating Gut Microbiota and Metabolic Changes.

Background: Spinal cord injury (SCI) damages the autonomic nervous system and affects the homeostasis of gut microbiota. Ursolic acid (UA) is a candidate drug for treating nervous system injury due to its neuroprotective and antioxidant functions. The purpose of our study was to investigate the role of UA on SCI and its mechanism. Methods: UA was administered to SCI mice and the solvent corn oil was used as control. The weight of the mice was recorded daily. Mice feces were collected 21 days after surgery for 16S rRNA-amplicon sequencing and untargeted metabolomics analysis. The expressions of NF-κB, IL-1ß, and TNF-α in the spinal cord and colon tissues of mice were detected by Western blot and Enzyme-linked immunosorbent assay, respectively. Immunohistochemistry was used to analyze the expression of NeuN, NF-200, and synapsin in the spinal cord tissues. Results: UA treatment increased body weight and soleus muscle weight of SCI mice. UA treatment inhibited inflammatory response and protected neuronal activity in SCI mice. UA improved the relative abundance of Muribaculaceae, Lachnospiraceae_NK4A136_group, and Alloprevotell genus in the gut tract of SCI mice. SCI destroyed the Glutamine_and_D-glutamate_metabolism, Nitrogen_metabolism, Aminoacyl-tRNA_biosynthesis, and Taurine_and_hypotaurine_metabolism in the gut of mice, which might be alleviated by UA. Conclusions: UA treatment could inhibit SCI progression by improving the gut environment and metabolic changes, promoting synaptic regeneration and anti-inflammatory effects.

The Impact of Diabetes on Vascular Disease: Progress from the Perspective of Epidemics and Treatments.

At present, the global incidence of diabetes has increased in countries with large populations, and the changes in developing regions are particularly worthy of attention. In the past 40 years or so, the income situation in China, India, and other countries has exploded, leading to changes in the way of life and work as well as an increase in the prevalence of diabetes. Metabolic disorders caused by diabetes can lead to secondary vascular complications, which have long-term malignant effects on the heart, kidneys, brain, and other vital organs of patients. Adequate primary prevention measures are needed to reduce the incidence of diabetic vascular complications, and more attention should be given to treatment after the disease. To this end, it is necessary to determine a standardized drug and physical therapy system and to build a more efficient and low-cost chronic disease management system.

Macrophage CD36 and TLR4 Cooperation Promotes Foam Cell Formation and VSMC Migration and Proliferation Under Circadian Oscillations.

Circadian rhythm disorders can accelerate atherosclerosis. This study aimed to determine the role of circadian disordered macrophages in atherosclerotic development. Mice were divided into NC group (normal circadian rhythm), L24 group (constant light), D12L12 group (weekly shift light/dark cycle), and D24 group (constant dark). Atherosclerotic progression was significantly accelerated in L24, D12L12, and D24 groups. Peritoneal macrophages from circadian disruption groups exhibited enhanced cytokine secretion and foam cell formation. Migration and proliferation of vascular smooth muscle cells (VSMCs) were increased under the conditioned medium of circadian disordered macrophages. The blockade of CD36 markedly inhibited foam cell formation. Compared with blocking CD36 or TLR4 alone, the co-inhibition of CD36 and TLR4 in macrophages further reduced cytokine secretion and more effectively inhibited VSMC migration and proliferation. In conclusion, the activation of CD36 and TLR4 in circadian disordered macrophages promotes foam cell formation and cytokine secretion and enhances VSMC migration and proliferation. Circadian rhythm disorders promote lipid uptake and cytokine secretion of macrophages by regulating CD36 and TLR4, and enhance VSMC migration and proliferation through the paracrine effect of macrophages.

Gut Microbiota Disorders Promote Inflammation and Aggravate Spinal Cord Injury Through the TLR4/MyD88 Signaling Pathway.

Background: In spinal cord injury (SCI), systemic inflammation and the death of nerve cells in the spinal cord are life threatening. The connection between gut microbiota and signaling pathways has been a hot research topic in recent years. The Toll-like receptor 4/Myeloid differentiation factor 88 (TLR4/MyD88) signaling pathway is closely related to the inflammatory response. This study explored whether the gut microbiota imbalance could affect the TLR4/MyD88 signaling pathway to regulate SCI to provide a new basis for SCI research and treatment. Methods: An SCI model was constructed to study the influence on the injury of gut microbiota. 16S amplicon sequencing was used to identify the diversity and abundance of gut microbes. Fecal microbiota transplantation was performed in mice with SCI. ELISA was used to detect the serum levels of pro-inflammatory and anti-inflammatory factors in mice. Hematoxylin and eosin staining was used to observe SCI in mice. Immunofluorescence was used to detect the rates of loss glial fibrillary acidic protein (GFAP), neuronal nuclear protein (NeuN), and ionized calcium-binding adapter molecule 1 (IBA1) in the spinal cord as indicators of apoptosis. The expression of the TLR4/MyD88 signaling pathway was detected by qRT-PCR and western blotting. Results: Significant differences were observed in the gut microbiota of SCI mice and normal mice. The gut microbiota of SCI mice was imbalanced. The levels of pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in SCI mice were increased, as was the level of the toxic induced nitric oxide synthase. The levels of anti-inflammatory factors IL-4, transforming growth factor-ß, and IL-10 were decreased, as was the level of arginase-1. The apoptosis rates of GFAP, NeuN, and IBA1 were increased. The TLR4/MyD88 signaling pathway was activated. In the SCI group, inflammation increased after fecal transplantation, apoptosis of GFAP, NeuN, and IBA1 increased, and SCI was more serious. Conclusion: The TLR4/MyD88 signaling pathway promotes the death of nerve cells by inducing inflammation. Gut microbiota dysregulation can lead to aggravated SCI by activating the TLR4/MyD88 signaling pathway.

Eucommia ulmoides Polysaccharides Attenuate Rabbit Osteoarthritis by Regulating the Function of Macrophages.

Background and purpose: Eucommia ulmoides polysaccharides (EUP) can regulate the immunity of macrophages, but the functional status of macrophages is related to osteoarthritis and synovial inflammation. The purpose of this study is to explore whether EUP has the effect of inhibiting osteoarthritis and its possible mechanism. Methods: MTT test was used to evaluate the appropriate concentration of EUP and real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the effect of EUP on gene expression in RAW 264.7 cells. The osteoarthritis model was constructed by the anterior cruciate ligament transection (ACLT) in the rabbits. These rabbits were divided into three groups, sham operation group, OA group, and EUP group. The changes in articular cartilage were detected by gross observation and histological staining, and Micro-CT tested subchondral bone. Finally, the changes of macrophages in synovial tissue were studied by immunohistochemistry. Results: The results showed that EUP at the concentration of 50ug/mL and 100ug/mL were beneficial to the proliferation of macrophages. The qPCR results indicated that EUP inhibited the expression of inflammation-related genes IL-6, IL-18 and IL-1ß, and promoted the expression of osteogenic and cartilage-related genes BMP-6, Arg-1 and transforming growth factor beta (TGF-ß). The results of in vivo experiments suggested that the degree of destruction of articular cartilage in the EUP group was significantly reduced, and the Osteoarthritis Research Society International (OARSI) score was significantly reduced. Compared with the OA group, the subchondral cancellous bone density of the EUP group increased, the number and thickness of trabecular bone increased, and the separation of trabecular bone decreased. Synovial macrophage immunohistochemistry results manifested that EUP, on the one hand, reduced M1 polarized macrophages, on the other hand, accumulated M2 polarized macrophages. Conclusion: EUP can promote articular cartilage repair and subchondral bone reconstruction. The regulation of the polarization state of macrophages may be one of its mechanisms to delay the progression of osteoarthritis.

Fish consumption, long-chain omega-3 fatty acids intake and risk of stroke: An updated systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: Although fish consumption or omega-3 intake is associated with cardio- cerebrovascular disease including stroke, their correlation is still controversial. Therefore, this meta-analysis is to identify the relationship between the risk of stroke and fish consumption or omega-3 intake. METHODS AND STUDY DESIGN: We searched the PubMed, EMBASE and Cochrane Library databases as of May 2019. Multivariateadjusted risk ratios (RRs) with 95% confidence interval (CI) for stroke in different level intake of fish or Longchain omega-3 polyunsaturated fatty acids (LC ω3-PUFAs) were pooled using a random-effects meta-analysis. A dose-response analysis was conducted with the 2-stage generalized least-squares trend program. RESULTS: Our meta-analysis identified a total of 17 prospective cohort studies including 14986 strokes events in 672711 individuals. Meta-analysis revealed that the higher fish consumption was significantly associated with lower risk of stroke (RR=0.871, 95% CI: 0.779-0.975, p=0.016), especially with ischemic stroke (RR=0.808, 95% CI: 0.696- 0.937, p=0.005). Meantime, the combined RR of total stroke was 0.859 (95% CI: 0.769-0.959, p=0.007) for the highest versus lowest intake of LC ω3-PUFAs, and stratification analysis showed that higher LC ω3-PUFAs intake was associated with reduced stroke risk in women (RR=0.793, 95% CI: 0.706-0.891, p=0.000) but not in men. In addition, the dose-response analysis showed fish consumption with 1000g per month and LC ω3-PUFAs intake with 0.5g per month was associated with 17.3% (RR=0.927, 95% CI: 0.83-0.98) and 14% (RR=0.86, 95% CI: 0.78-0.95) lower risk of stroke, respectively. CONCLUSIONS: Both fish consumption and LC ω3-PUFAs intake were negatively associated with the risk of stroke, especially in women, which suggest that increased intake of fishery products and LC ω3-PUFAs may benefit primary prevention of stroke.

CML/RAGE Signal Bridges a Common Pathogenesis Between Atherosclerosis and Non-alcoholic Fatty Liver.

Non-alcoholic fatty liver disease (NAFLD) has become a common chronic disease in the world. NAFLD is not only a simple intrahepatic lesion, but also affects the occurrence of a variety of extrahepatic complications. In particular, cardiovascular complications are particularly serious, which is the main cause of death in patients with NAFLD. To study the relationship between NAFLD and AS may be a new way to improve the quality of life in patients with NAFLD. As we all known, inflammatory response plays an important role in the occurrence and development of NAFLD and AS. In this study, we found that the accumulation of Nε-carboxymethyllysine (CML) in the liver leads to hepatic steatosis. CML can induce the expression of interleukin (IL-1ß), interleukin (IL-6), tumor necrosis factor (TNF-α), C-reactionprotein (CRP) by binding with advanced glycosylation end-product receptor (RAGE) and accelerate the development of AS. After silencing RAGE expression, the expression of pro-inflammatory cytokines was inhibited and liver and aorta pathological changes were relieved. In conclusion, CML/RAGE signal promotes the progression of non-alcoholic fatty liver disease and atherosclerosis. We hope to provide new ideas for the study of liver vascular dialogue in multi organ communication.

Role of Sortilin and Matrix Vesicles in Nε-Carboxymethyl-Lysine-Induced Diabetic Atherosclerotic Calcification.

BACKGROUND AND AIMS: To investigate the role of Sortilin and matrix vesicles (MVs) in Nε-Carboxymethyl-lysine (CML)-induced diabetic atherosclerotic calcification (AC). METHODS: At human level, the correlation between Sortilin and CD9 (marker proteins of MVs) in serum MVs and CML in serum was explored by enzyme-linked immunosorbent assay (ELISA) detection and Pearson correlation analysis. After a diabetic apoE-/- mouse model was constructed, the calcification of aorta and the expressions of related proteins under CML and MVs injection were observed by calcification staining, immunofluorescence staining, and Western blot. MVs levels released by smooth muscle cells (SMCs) under different treatments was detected by nanometer tracking analysis (NTA). After treating SMCs with MVs and Anti-Sortilin, cell calcification was observed by Alizarin red staining. RESULTS: Serological analysis of patients showed that the concentrations of Sortilin and CD9 in serum MVs were positively correlated with the concentration of CML in serum. Animal experiments showed that CML could promote the progression of diabetic AC and the high expression of Sortilin in plaques. Diabetic apoE-/- mouse tail vein injection of CML-induced SMCs-derived MVs obviously aggravated AC. Cell experiment results showed that a high concentration of CML significantly promoted the release of MVs from SMCs. MVs from this source could markedly worsen cell calcification, while the administration of GW4869 (a widely used extracellular vesicles biogenesis inhibitor) significantly reduced cell calcification. Finally, treatment of high concentrations of CML could also promote the recruitment of Sortilin to MVs, and administration of Anti-Sortilin could markedly reduce cell calcification caused by MVs. CONCLUSION: We proved that CML not only affects the release of MVs from SMCs but also affects the recruitment of Sortilin to MVs, thereby promoting diabetic AC. This discovery may provide a new strategy for targeted prevention of vascular calcification in diabetes.

Healing pattern classification for thoracolumbar burst fractures after posterior short-segment fixation.

BACKGROUND: Thoracolumbar burst fractures can be treated with posterior short-segment fixation. However, no classification can help to estimate whether the healed vertebral body will have sufficient stability after implant removal. We aimed to develop a Healing Pattern Classification (HPC) to evaluate the stability of the healed vertebra based on cavity size and location. METHODS: Fifty-two thoracolumbar burst fracture patients treated with posterior short-segmental fixation without fusion and followed up for an average of 3.2 years were retrospectively studied. The HPC was divided into 4 types: type I - no cavity; type II - a small cavity with or without the violation of one endplate; type III - a large cavity with or without the violation of one endplate; and type IV - a burst cavity with the violation of both endplates or the lateral cortical shell. The intraobserver and interobserver intraclass correlation coefficients (ICCs) of the HPC were assessed. The demographic characteristics and clinical outcomes of the cohort were compared between the stable group (types I and II) and the unstable group (types III and IV). Logistic regression was conducted to evaluate risk factors for unstable healing. RESULTS: The intraobserver and interobserver ICCs of the HPC were 0.86 (95% CI = 0.74-0.90) and 0.77 (95% CI = 0.59-0.86), respectively. While the unstable healing group (types III and IV) accounted for 59.6% of the patients, most of these patients were asymptomatic. The preoperative Load Sharing Classification (LSC) comminution score may predict the occurrence of unstable healing (OR = 8.4, 95% CI = 2.4-29.7). CONCLUSIONS: A reliable classification for assessing the stability of a healed vertebra was developed. With type I and II healing, the vertebra is considered stable, and the implant can be removed. With type III healing, the vertebra may have healing potential, but the implant should not be removed unless type II healing is achieved. With type IV healing, the vertebra is considered extremely unstable, and instrumentation should be maintained. Assessing the LSC comminution score preoperatively may help to predict unstable healing after surgery.

Human glioma stem-like cells induce malignant transformation of bone marrow mesenchymal stem cells by activating TERT expression.

We investigated whether glioma stem-like cells (GSCs) malignantly transformed bone marrow mesenchymal stem cells (tBMSCs) in the tumor microenvironment. Transplantation of enhanced green fluorescence protein (EGFP)-labeled BMSCs into irradiated athymic nude mice was followed by intracranial injection of red fluorescent protein-expressing glioma stem-like cells (SU3-RFP-GSCs). Singly cloned EGFP-BMSCs, harvested from the intracranial tumors showed TERT overexpression, high proliferation, colony formation and invasiveness in Transwell matrigel assays. Transfection of normal BMSCs with TERT (TERT-BMSCs) enhanced proliferation, colony formation and invasiveness, though these characteristics remained lower than in tBMSCs. The tBMSCs and TERT-BMSCs showed high surface expression of CD44, CD105, CD29 and CD90 and an absence of CD31, CD34, CD45, and CD11b, as in normal BMSCs. Alizarin red S and oil red O staining confirmed tBMSCs and TERT-BMSCs transdifferentiated into osteocytes and adipocytes, respectively. When normal BMSCs were indirectly co-cultured in medium from SU3-RFP-GSCs, they exhibited increased growth and proliferation, suggesting paracrine factors from GSCs induced their malignant transformation. Tumorigenicity assays in athymic nude mice showed that transplanted tBMSCs and TERT-BMSCs generated 100% and 20% subcutaneous tumors, respectively, while normal BMSCs generated no tumors. GSCs thus induce malignant transformation of BMSCs by activating TERT expression in BMSCs.

Enhancement of DEN-induced liver tumourigenesis in hepatocyte-specific Lass2-knockout mice coincident with upregulation of the TGF-ß1-Smad4-PAI-1 axis.

Longevity assurance homolog 2 of yeast LAG1 (Lass2) gene is capable of suppressing the proliferation and metastasis of several types of tumours including liver cancer. In the present study, hepatocyte-specific Lass2-knockout (Lass2 KO) and wild-type (WT) mice were exposed to the carcinogen, diethylnitrosamine (DEN), to induced liver tumours. At week 23 following DEN injection, tumours were produced in 100% of the Lass2 KO mice and 21.4% of the WT mice. At week 40, 100% of the Lass2 KO mice and 78.6% of the WT mice developed tumours, with no distinct significant difference in tumour occurrences between the two genotypes; yet, tumours in the Lass2 KO mouse livers were more numerous and larger in size. Hepatocellular carcinoma (HCC) was confirmed by α-fetoprotein (AFP). PCNA and EdU assays indicated more active proliferation whereas TUNEL assay revealed decreased apoptosis in Lass2 KO livers, when compared with the WT control. The expression of plasminogen activator inhibitor type-1 (PAI-1), a tumour-promoting gene, in the liver tissues of the 2 genotypes was detected using qPCR and western blotting, showing that PAI-1 levels were significantly elevated in Lass2 KO livers at week 40 following DEN introduction. Moreover, the expression of PAI-1-related TGF-ß1, Smad-4 and -7 was detected, displaying an elevation in TGF-ß1 and Smad-4 (not including Smad-7) in the Lass2 KO livers. Our data demonstrates that i) Lass2 is a protective gene against DEN-induced liver tumourigenesis; and ii) upregulation of the TGF-ß1-Smad4-PAI-1 axis may contribute to the vulnerability of Lass2-knockout mice to DEN.

The mTOR inhibitor AZD8055 inhibits proliferation and glycolysis in cervical cancer cells.

The aim of the present study was to determine the effect of AZD8055 on proliferation, apoptosis and glycolysis in the human cervical cancer cell line HeLa and to investigate the underlying mechanism(s) of action. HeLa human cervical cancer cells were treated with 10 nM AZD8055 for 24, 48 or 72 h. MTT was used to determine cell proliferation. Annexin V/propidium iodide staining was used to determine cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. RNA and protein expression were examined by qRT-PCR and western blotting, respectively. Treatment with AZD8055 inhibited proliferation and glycolysis, and induced apoptosis in HeLa cells in a time-dependent manner. During the prolonged treatment with AZD8055, the phosphorylation of mammalian target of rapamycin (mTOR) C1 substrates p70S6K and phosphorylation of the mTORC2 substrate Akt were deregulated, suggesting that the activity of mTOR was downregulated. Furthermore, our study showed that the expression of miR-143 was upregulated in a time-dependent manner in HeLa cells treated with AZD8055. In summary, the present study reveals a novel antitumor mechanism of AZD8055 in HeLa human cervical cancer cells.