Lactate-upregulated NADPH-dependent NOX4 expression via HCAR1/PI3K pathway contributes to ROS-induced osteoarthritis chondrocyte damage.

Increasing evidence shows that metabolic factors are involved in the pathological process of osteoarthritis (OA). Lactate has been shown to contribute to the onset and progression of diseases. While whether lactate is involved in the pathogenesis of OA through impaired chondrocyte function and its mechanism remains unclear. This study confirmed that serum lactate levels were elevated in OA patients compared to healthy controls and were positively correlated with synovial fluid lactate levels, which were also correlated with fasting blood glucose, high-density lipoprotein, triglyceride. Lactate treatment could up-regulate expressions of the lactate receptor hydroxy-carboxylic acid receptor 1 (HCAR1) and lactate transporters in human chondrocytes. We demonstrated the dual role of lactate, which as a metabolite increased NADPH levels by shunting glucose metabolism to the pentose phosphate pathway, and as a signaling molecule up-regulated NADPH oxidase 4 (NOX4) via activating PI3K/Akt signaling pathway through receptor HCAR1. Particularly, lactate could promote reactive oxygen species (ROS) generation and chondrocyte damage, which was attenuated by pre-treatment with the NOX4 inhibitor GLX351322. We also confirmed that lactate could increase expression of catabolic enzymes (MMP-3/13, ADAMTS-4), reduce the synthesis of type II collagen, promote expression of inflammatory cytokines (IL-6, CCL-3/4), and induce cellular hypertrophy and aging in chondrocytes. Subsequently, we showed that chondrocyte damage mediated by lactate could be reversed by pre-treatment with N-Acetyl-l-cysteine (NAC, ROS scavenger). Finally, we further verified in vivo that intra-articular injection of lactate in Sprague Dawley (SD) rat models could damage cartilage and exacerbate the progression of OA models that could be countered by the NOX4 inhibitor GLX351322. Our study highlights the involvement of lactate as a metabolic factor in the OA process, providing a theoretical basis for potential metabolic therapies of OA in the future.

Brevilin A Inhibits VEGF-Induced Angiogenesis through ROS-Dependent Mitochondrial Dysfunction.

Brevilin A (BA), a sesquiterpene lactone isolated from Centipeda minima herb, has been identified to exhibit potent anticancer activity. However, the potential pharmacological effect and mechanism of BA in regulating endothelial cell (EC) angiogenesis, a key event in tumor growth, is poorly understood. In this study, BA was shown to significantly prevent vascular endothelial growth factor (VEGF) induced EC angiogenic capacities in vitro, ex vivo, and in vivo. Subsequent functional assays revealed that BA dose dependently inhibited VEGF-stimulated survival, proliferation, migration, and triggered apoptosis activity in human umbilical vein endothelial cells (HUVECs), as well as suppressed the expression of antiapoptotic protein Bcl-2, increased the expression of proapoptotic protein caspase-3 and Bax, and suppressed PI3K/AKT pathway. Meanwhile, BA was also able to depolarize mitochondrial membranal permeability (MMP), accelerate mitochondrial superoxide accumulation, induce intracellular reactive oxygen species (ROS) production, and decreased intracellular glutathione (GSH) in HUVECs. Furthermore, both mitochondria-specific superoxide scavenger Mito-TEMPOL and broad-spectrum antioxidant N-acetyl-cysteine (NAC) dramatically abolished BA-induced mitochondrial dysfunction and mitochondrial ROS production, causing the reversion of PI3K/AKT pathway and repression of apoptosis, eventually correcting the impaired endothelial behavior in survival, growth, migration, and angiogenesis. Collectively, our data for the first time identified a new mechanism for antiangiogenic effect of BA in vascular EC, one that is based on the regulation of mitochondrial-dependent ROS overproduction.

Taraxasterol prompted the anti-tumor effect in mice burden hepatocellular carcinoma by regulating T lymphocytes.

Hepatocellular carcinoma (HCC) is a common digestive malignant tumor with high morbidity and mortality worldwide, however, the treatment of HCC and prognosis of patients are not optimistic, finding more effective treatments are imperative. Taraxacum officinale (L.) Weber ex F.H.Wigg is a perennial herb of compositae, and our study has demonstrated that Taraxacum officinale polysaccharide has certain anti-tumor effect on HCC cells. Taraxasterol (TS) is a natural product extracted from Taraxacum officinale with strong physiological, pharmacological and biological activities, but the effect of TS on HCC is yet to be determined. Therefore, the aim of this study is to explore the effect of dandelion sterol on HCC in vivo and in vitro. The results showed that TS significantly inhibited the proliferation, induced apoptosis and blocked cell cycle in HCC cell lines HepG2 and Huh7 cells in vitro. TS inhibited the tumor growth of H22 bearing mice and the expression of Ki67 in vivo. More importantly, TS regulated the immunity of H22 bearing mice by elevating the ratio of CD4+ T cells in spleen, and increasing the number of T cell infiltration in tumor tissue. Except immunomodulation, the mechanism of tumor growth inhibition may be related to the regulation of apoptosis related proteins and IL-6/STAT3 pathway. TS significantly inhibited the growth of HCC cells both in vitro and in vivo. The study would provide a theoretical basis for the new application of TS and the adjuvant treatment of malignant tumor with traditional Chinese medicine.

MicroRNA-520c-3p targeting of RelA/p65 suppresses atherosclerotic plaque formation.

Atherosclerosis is a chronic inflammatory disease, and it's the leading cause of death worldwide. Dysregulation of microRNAs (miRNAs) has been found to be associated with atherosclerosis. miR-520c-3p has been implicated in several types of cancer. However, little is known about the role of miR-520c-3p in atherosclerosis. In this study, we found that miR-520c-3p agomir decreased atherosclerotic plaque size, collagen content, the quantity of PCNA-positive cell and RelA/p65 expression of vascular smooth muscle cells (VSMCs) in the aortic valve of apoE-/- mice in vivo. The possible mechanisms of the protective effects of miR-520c-3p on atherosclerotic mice were then investigated in VSMCs. in vitro experiments showed that miR-520c-3p expressions were significantly reduced in human aortic vascular smooth muscle cell (HASMCs) treated with platelet-derived growth factor (PDGF-BB). miR-520c-3p mimics repress PDGF-BB-mediated the proliferation, migration and decrease in the percentage of cells in G2/M phase, which was associated with downregulation of RelA/p65. Mechanistically, miRNA pull-down, luciferase reporter and mRNA stability assays confirmed miR-520c-3p mimics was able to directly target 3'-UTR of RelA/p65 mRNA and decreased half-life of RelA/p65 mRNA in HASMCs. Overexpression of RelA/p65 reversed the inhibition of cell proliferation induced by miR-520c-3p mimics in HASMCs. In conclusion, our findings suggest that miR-520c-3p inhibits PDGF-BB-mediated the proliferation and migration of HASMCs by targeting RelA/p65, which may provide potential therapeutic strategies in atherosclerosis treatment.

Impact of Wortmannilactone F and G31P on Clonorchis Sinensis-infected mice.

Clonorchis sinensis could induce inflammation, epithelial hyperplasia and fibrosis in the intrahepatic bile duct as a food-borne parasite, which was associated with the development of cholangiocarcinoma (CCA). Praziquantel was the most effective drug on treatment of this kind of parasite. However, new drugs with minimal toxicity to the host were urgently needed due to the side effects of Praziquantel and its CCA risk. In this study, helminth mitochondria respiratory chain blocker Wortmannilatone F (WF) and IL-8 analogue CXCL8 (3-72) K11R/G31P were used to treat BALB/C mice infected by Clonorchis sinensis. We investigated the gross and histopathological morphology of the liver, inflammation-associated cytokine IL-6, lipid peroxidation-related proteins cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), collagen fiber accumulation and fibroblast-specific protein 1 (FSP1), malignant markers proliferating cell nuclear antigen (PCNA) and cytokeratin 19 (CK19), as well as the disinfection effect on these parasites in vitro. WF inhibited and killed the worms dramatically, and the combination of WF with G31P improved the condition of the hepatobiliary duct tissue greatly. These outcomes indicated that the combination of WF and G31P was a potential therapeutic method to treat the Clonorchis sinensis infection.

Effects of Mycobacterium tuberculosis Rv1096 on mycobacterial cell division and modulation on macrophages.

Mycobacterium tuberculosis is capable of escaping the clearance of immune system mainly due to its complex constituents of cell wall. Certain studies show that glycoproteins are involved in immune evasion and act as virulence factors. Peptidoglycan deacetylase Rv1096 is a member of mannosylated proteins. Previously, we reported Rv1096 protein contributed to the resistance of Mycobacterium smegmatis (M. smegmatis) to lysozyme, but more characterization of this protein is required where further intracellular function is unknown. Here, Rv1096 was heterologously over-expressed in the fast-growing and nonpathogenic M. smegmatis (named as M. smegmatis/Rv1096). We observed the morphological alterations in M. smegmatis/Rv1096 including an elongated rod-like shape and increased amounts of Z-rings, which implied that Rv1096 facilitated the cell growth and division. Moreover, a series of assays concerning the interaction between M. smegmatis/Rv1096 and host were carried out. The results showed that M. smegmatis/Rv1096 evaded the killing of macrophages due to the inhibition of phagosome-lysosome fusion, nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. The secretion of interleukin-12 and tumor necrosis factor-α was also impaired by Rv1096. In addition, five putative interaction partners of Rv1096 were identified, which possibly cooperated with Rv1096 in cell division and immune regulation. These results suggested that Rv1096 had effects on mycobacterial division and might act as a virulence factor to mediate the immune evasion in macrophage during mycobacterial infection.

Killing the muscular larvae of Trichinella spiralis and the anti-fibrotic effect of the combination of Wortmannilatone F and recombinant G31P in a murine model of trichinellosis.

Although trichinosis is one of the global food-borne parasitic diseases and is considered an emerging/re-emerging disease that has been reported in 66 countries, the drugs for its prevention and treatment have not been thoroughly investigated. Wortmannilactone F (WF) has been reported as a blocker of the helminth mitochondria respiratory chain by inhibiting NADH-fumarate reductase in the mitochondrial inner membrane. CXCL8 (3-73) K11R/G31 P(G31 P) has been reported as a CXCL8 analogue that has the affinity to CXCR1 and CXCR2. Male BALB/c mice were orally fed with 150 infective Trichinella spiralis (T. spiralis) larvae. Then, T. spiralis-infected mice were treated with WF and G31 P. The number and morphological analysis of encapsulated T. spiralis, collagen fiber accumulation, and the expression of angiogenic factors were investigated. WF and G31 P dramatically decreased the numbers of encapsulation, decreased collagen fibers, and suppressed angiogenesis. These findings indicate that the combination of WF and G31 P is a potential therapeutic strategy of Trichinellosis.

Curcumin sensitizes lymphoma cells to DNA damage agents through regulating Rad51-dependent homologous recombination.

Curcumin is a natural compound isolated from the rhizome of Curcuma longa. It possesses anti-tumor activity through arresting cell cycles and promoting cell apoptosis. However, the effect of curcumin on DNA damage is not well defined. In this study, we investigated the effect of curcumin on inducing DNA damage and on sensitizing lymphoma cells to anti-tumoral DNA damage drugs. Western blot showed curcumin induced γ-H2AX foci in CH12F3 lymphoma cells, which suggests curcumin induces DNA breaks. In addition, curcumin decreased the expression of Rad51, which suggests curcumin induces DNA damage through regulating Rad51-dependant homologous recombination. Rad51-dependant homologous recombination is a vital DNA repair pathway for cancer cells to resist anti-tumoral DNA damage drugs, therefore, we studied the effect of curcumin on the sensitizing lymphoma cells to various chemotherapeutic drugs. We found low level of curcumin (5µM) sensitized lymphoma cells to anti-tumoral DNA damage agents including cisplatin, methyl methanesulfonate, hydroxyurea and camptothecin. We also found curcumin sensitized CH12F3 lymphoma cells to DNA-PK and PARP inhibitors. Flow cytometry analysis showed curcumin promoted apoptosis and western blot analysis confirmed curcumin activated caspase3-dependent apoptosis. Taken together, these results demonstrate that curcumin induces DNA damage through regulating Rad51-dependant homologous recombination and triggers caspase3-dependent apoptosis, more importantly, curcumin sensitizes lymphoma cells to various DNA damage drugs. Consequently, curcumin would be a potent agent for sensitizing lymphoma cells to anti-tumoral chemotherapeutic agents.

Evaluation of recombinant CXCL8(3-73)K11R/G31P in muscle fibrosis and Trichinella larvae encapsulation in a murine model of trichinellosis.

Trichinella spiralis (T. spiralis) larvae in raw or inadequately cooked meat can cause chronic infections in a wide range of hosts including humans. During the development inside the skeletal muscles, T. spiralis larvae infect muscle cells accompanying with the infiltration of host inflammatory cells, eventually create a new type of cell known as nurse cell developing a surrounding vascular network to support the larvae development. Controlling of host inflammatory responses and angiogenesis influences both the nurse cell differentiation and the parasite larvae development. CXCL8 is a chemokine that acts on G-protein coupled receptors, of which activation contributes to fibrosis and angiogenesis. CXCL8(3-73)K11R/G31P (G31P) has been reported as a CXCL8 analogue. The aim of this study is to investigate the effect of G31P in inflammatory responses and the development of T. spiralis larvae in muscle tissues of mice infected with T. spiralis. The level of inflammatory factors and the morphology of T. spiralis larvae in infected tissues were investigated through ELISA and electron-microscopy analysis. G31P up-regulated IFN-γ and down-regulated CXCL8 level, and impaired the encapsulation of T. spiralis larvae in vivo. The results showed that G31P influenced the development of T. spiralis larvae in muscle tissues.

Platelet-derived growth factor-BB induces matrix metalloproteinase-2 expression and rat vascular smooth muscle cell migration via ROCK and ERK/p38 MAPK pathways.

Matrix metalloproteinases (MMP) play a pivotal role in the pathogenesis of cardiovascular diseases. Their expressions are altered in response to a variety of stimuli, including growth factors, inflammatory markers, and cytokines. In this study, we demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces a dose- and time-dependent increase in MMP-2 expression in rat vascular smooth muscle cells (VSMC). Treatment with either the Rho-associated protein kinase (ROCK) inhibitor Y-27632 or suppression of ROCK-1/2 by small interfering RNA technology significantly reduced the MMP-2 expression, thus suggesting that ROCK regulates such expression. Similar results were observed when VSMC were pretreated with either U0126 or SB203580, which are selective inhibitors of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, respectively, thus suggesting that these kinases are important for the induction of MMP-2 expression by PDGF-BB. In conclusion, these results described a novel mechanism in atherosclerosis through PDGF-BB signaling in VSMC, in which MMP-2 expression is induced via extracellular signal-regulated kinases and p38 mitogen-activated protein kinase phosphorylation, as well as ROCK.

Anisakiasis in China: the first clinical case report.

Anisakiasis is a parasitic disease acquired by humans when ingesting raw or undercooked fish infected with L3 larvae of the nematode genus Anisakis or Pseudoterranova. Here we report the first case of human anisakiasis in China. The patient, male, 56 years old, Dalian citizen, was admitted into the hospital with vomiting, peripheral umbilicus and abdominal distension, and frequent mucous diarrhea. The patient was examined using an electronic gastroscope, which displayed a parasite residing in the stomach, and subsequently gastroscope-assisted surgery was implemented. A white round worm was removed from the patient and stained. It was identified as L3 larvae of Anisakis. After the removal of the L3 larvae of Anisakis, the inflammation symptoms disappeared. As the first report of clinical case of Anisakis infection in China, the morphology of L3 Anisakis larvae from the patient is described and discussed. We conclude that anisakiasis should be considered in patients who have a habit of eating raw fish and who display associated symptoms.

Rho-associated protein kinase isoforms stimulate proliferation of vascular smooth muscle cells through ERK and induction of cyclin D1 and PCNA.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) plays an important role in vascular diseases. The Rho-associated protein kinase (ROCK) signaling pathway is now well recognized for its role in VSMC migration and proliferation. Recently, a number of studies revealed that different isoforms of ROCK have distinct functions in VSMCs. We have reported that ROCK1, rather than ROCK2, induces platelet-derived growth factor (PDGF)-BB-stimulated migration of VSMCs. In the current study, we aimed to investigate the roles of ROCK1/2 in PDGF-induced rat aorta VSMC proliferation by manipulating ROCK gene expression. The results revealed that knock-down of both ROCK1 and ROCK2 by siRNA technology decreased PDGF-BB-generated VSMC proliferation by inhibiting the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1. In addition, up-regulation of ROCK1 expression through transfection, further increased the proliferation of VSMCs induced by PDGF-BB. The ERK inhibitor U0126 reduced the proliferation and expression of PCNA and cyclinD1, and ROCK1 and ROCK2 siRNA decreased the level of ERK in the nucleus. These results demonstrated that ROCK1 and ROCK2 could promote VSMC proliferation through ERK nuclear translocation, regulating the expression of PCNA and cyclin D1 protein.

[Effects of Fructus Psoralea and Brucea javanica on the level of IL-2 and NK cell in rats infected with Pneumocystis carinii].

SD rat model of PCP was established by subcutaneous injection with dexamethasone. The treatment groups received Fructus Psoralea (FP, 10.0 mg/kg), Brucea javanica (BJ, 1.2 mg/kg) and a mixture of the two Chinese herbs(FP 5mg/kg, BJ 0.6 mg/kg) respectively. By means of detecting the level of IL-2 in sera and NK cells in spleens, the effect of FP and BJ on the level of IL-2 and NK cells in rats with Pneumocystis carinii pneumonia (PCP) was observed, with SMZco treatment group (TMP 50 mg/kg, SMZ 250 mg/kg) and groups of infected and normal rats as controls. Compared with the infected group, the level of IL-2(526.1 +/- 5.5) pg/ml and NK cells (27.1% +/- 0.8%) significantly increased in the FP group (P < 0.01), followed by the FP/BJ combination group [(314.7 +/- 6.7) pg/ml, 22.9% +/- 0.9%) (P < 0.05)], and BJ group [(285.4 +/- 6.1) pg/ml, 20.7% +/- l.0%) (P < 0.05)]. Chinese herbs Fructus Psoralea and Brucea javanica show an immune regulatory action on the PCP rats.

[Immunological regulation and treatment of Brucea javanica and Fructus Psoraleae on rats with Pneumocystis carinii pneumonia].

OBJECTIVE: To study the immunological regulation and treatment of Brucea javanica and Fructus Psoraleae, traditional Chinese medicine, on rats with Pneumocystis carinii pneumonia (PCP). METHODS: Rats were injected subcutaneously by dexamethasone. When the rats got Pc infected, they were divided into two groups: rats in one group were treated with the mixture of Brucea javanica and Fructus Psoraleae and another group was used as infected control. Control with normal rats was also established. Observations were made on the number of cysts in lungs and the changes of CD4+ T cells, CD8+ T cells and TNF-alpha in the serum to demonstrate the immunological regulation and killing effect of the medicine on cysts in the infected rats. RESULTS: The body weight of the rats treated with Brucea javanica and Fructus Psoraleae increased considerably than that of immunosuppressed rats and the normal control. The damaged lung got improved and repaired, and a significant cyst reduction was shown in the treated group. The CD4+ T cells, CD8+ T cells and level of TNF-alpha in serum also increased in the treated group significantly. CONCLUSION: The mixture of Brucea javanica and fructus psoraleae plays an immunological regulation on rats with Pneumocystis carinii pneumonia and shows certain killing effect on the cysts.

[Electron microscopical observation on rats with Pneumocystis carinii pneumonia treated with Brucea javanica and Fructus Psoraleae].

Rats with Pneumocystis carinii pneumonia (PCP) were established by hypodermic injection of dexamethasone and treated by Brucea javanica combined with Fructus Psoraleae 2 ml (Brucea javanica 0.12 mg and Fructus Psoraleae 1.0 mg) per rat per day for 7 days or 14 days. The effect of cyst-killing and the pathological change were observed under transmission electron microscope. Lung damage was alleviated or repaired, and a significant reduction of cysts was shown in the treatment group. The results show that a combination of Brucea javanica and Fructus Psoraleae played a significant restraining and killing effect on cysts, and helped repair the impairment of pneumonia.