Ginsenoside Rb1 prevents age-related endothelial senescence by modulating SIRT1/caveolin-1/enos signaling pathway.

Background: Advancing age is one of the independent risk factors for cardiovascular disorders. The Compendium of Materia Medica, a classic book on traditional Chinese medicine, states that ginseng "harmonizes the five internal organs, calming the spirit and prolonging the years of life." Considered one of the primary bioactive compounds derived from Panax ginseng, ginsenoside Rb1 (g-Rb1) has been scientifically suggested to possess anti-senescence efficacy. More research is needed to explore the vascular pharmacological activity and potential clinical application value of g-Rb1. Aims of the study: Our previous study demonstrated that g-Rb1 could mitigate cellular senescence via the SIRT1/eNOS pathway. This study was performed to explore the exact mechanisms by which g-Rb1 modulates the SIRT1/eNOS pathway. Materials and methods: We used human primary umbilical vein endothelial cells (HUVECs) to establish a replicative ageing model. Real-time (RT‒PCR), western blotting, small interfering RNA (siRNA), and immunoprecipitation were conducted to detect the effect of g-Rb1 on the SIRT1/caveolin-1/eNOS axis. Results: G-Rb1 increased NO production and alleviated replicative senescence of HUVECs. The application of g-Rb1 elevated the mRNA and protein abundance of both SIRT1 and eNOS while concomitantly suppressing the expression of caveolin-1. Inhibition of SIRT1 and eNOS by siRNAs suppressed the anti-senescence function of g-Rb1, while caveolin-1 siRNA could enhance it. G-Rb1 decreased the acetylation level of caveolin-1 and increased NO production, which was suppressed by SIRT1 siRNA. Both g-Rb1 and caveolin-1 siRNA could reduce the acetylation level of eNOS and increase NO production. Conclusion: G-Rb1 prevents age-related endothelial senescence by modulating the SIRT1/caveolin-1/eNOS signaling pathway.

Chemical characterization and effects of volatile oil of Alpinia zerumbet on the quality of collagen deposition and caveolin-1 expression in a muscular fibrosis murine model / Caracterização química e efeitos do óleo volátil de Alpinia zerumbet na qualidade da deposição do colágeno e expressão da caveolina-1 em modelo murino de fibrose muscular

Abstract This study evaluated the effect of the volatile oil of Alpinia zerumbet (VOAz) on caveolin-1 gene expression and muscular fibrosis. The rats were immobilized to induce fibrosis of the gastrocnemius muscle, and they were treated with VOAz. Collagen quality was assessed by histology and the expression of the caveolin-1 (CAV-1) gene was evaluated using qPCR. Histomorphological analysis indicated a significant reduction in the perimeter, width, and intensity of collagen in the treated groups, thus showing that the oil was effective in regulating the quality of collagen at the three concentrations. The results of expression levels suggested a decrease in the lesioned group and in two treatment groups (0.0115 µg/g and 0.009 µg/g). However, with the lowest concentration (0.0065 µg/g), no significant difference was observed, with levels similar to those found in healthy tissue. Therefore, the results showed that VOAz has the potential to be a non-invasive and low-cost alternative to aid in the treatment of muscular fibrosis.

Resumo Este estudo avaliou o efeito do óleo volátil de Alpinia zerumbet (OVAz) na expressão do gene da caveolina-1 e na fibrose muscular. Os ratos foram imobilizados para induzir a fibrose do músculo gastrocnêmio, e foram tratados com OVAz. A qualidade do colágeno foi avaliada com histologia e à expressão do gene caveolina-1 (CAV-1) foi avaliada usando qPCR. A análise histomorfológica indicou uma redução significativa no perímetro, largura e intensidade do colágeno nos grupos tratados. Os resultados dos níveis de expressão sugeriram diminuição nos grupos de lesão e em dois grupos de tratamento (0,0115 µg/g e 0,009 µg/g). No entanto, com a menor concentração (0,0065 µg/g), não foi observada diferença significativa, apresentando níveis semelhantes aos encontrados em tecido saudável. O uso do OVAz foi eficaz para reverter as alterações do colágeno causadas pela fibrose, e sua menor concentração apresentou uma possível tendência de aumento na expressão do CAV-1. Portanto, os resultados mostraram que o OVAz tem potencial para ser uma alternativa não invasiva e de baixo custo para auxiliar no tratamento da fibrose muscular.

Erchen decoction alleviates the progression of NAFLD by inhibiting lipid accumulation and iron overload through Caveolin-1 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: A combination of 6 different Chinese herbs known as Erchen decoction (ECD) has been traditionally used to treat digestive tract diseases and found to have a protective effect against nonalcoholic fatty liver disease (NAFLD). Despite its efficacy in treating NAFLD, the precise molecular mechanism by which Erchen Decoction regulated iron ion metabolism to prevent disease progression remained poorly understood. AIM OF STUDY: Our study attempted to confirm the specific mechanism of ECD in reducing lipid and iron in NAFLD from the perspective of regulating the expression of Caveolin-1 (Cav-1). STUDY DESIGN: In our study, the protective effect of ECD was investigated in Palmitic Acid + Oleic Acid-induced hepatocyte NAFLD model and high-fat diet-induced mice NAFLD model. To investigate the impact of Erchen Decoction (ECD) on lipid metabolism and iron metabolism via mediating Cav-1 in vitro, Cav-1 knockdown cell lines were established using lentivirus-mediated transfection techniques. MATERIALS AND METHODS: We constructed NAFLD model by feeding with high-fat diet for 12 weeks in vivo and Palmitic Acid + Oleic Acid treatment for 24 h in vitro. The regulation of Lipid and iron metabolism results by ECD were detected by serological diagnosis, immunofluorescent and immunohistochemical staining, and western blotting. The binding ability of 6 small molecules of ECD to Cav-1 was analyzed by molecular docking. RESULTS: We demonstrated that ECD alleviated the progression of NAFLD by inhibiting lipid accumulation, nitrogen oxygen stress, and iron accumulation in vivo and in vitro experiments. Furthermore, ECD inhibited lipid and iron accumulation in liver by up-regulating the expression of Cav-1, which indicated that Cav-1 was an important target for ECD to exert its curative effect. CONCLUSIONS: In summary, our study demonstrated that ECD alleviated the accumulation of lipid and iron in NAFLD through promoting the expression of Cav-1, and ECD might serve as a novel Cav-1 agonist to treat NAFLD.

Effect of electroacupuncture at "Jiaji" (EX-B2) on senescence of nucleus pulposus cells of degenerated lumbar intervertebral disc in rabbits. / 电针"夹脊"å¯¹è °æ¤Žé—´ç›˜é€€å˜å ”é€€å˜æ¤Žé—´ç›˜é«“æ ¸ç»†èƒžè¡°è€çš„å½±å“.

OBJECTIVES: To observe the effect of electroacupuncture (EA) at "Jiaji"(EX-B2) on body mass, motor function, expression of caveolin-1 (Cav-1) in nucleus pulposus cells and annulus fibrosus tissue, telomerase activi-ty, relative telomere length and different cell cycle ratio of nucleus pulposus cells in rabbits with intervertebral disc degeneration(IVDD), so as to investigate its mechanism underlying delaying senescence of the degenerated lumbar intervertebral disc nucleus pulposus cells. METHODS: Twenty-five male New Zealand rabbits with mature bones were divided into control, sham operation, model, EA, and acupuncture groups, with 5 rabbits in each group. The IVDD model was established by inserting kirschner wires to the vertebral bone surface between the lumbar (L)4 and L5 vertebrae, followed by applying continuous axial pressure for 28 d. EA (2 Hz/15 Hz, 1-2 mA) or acupuncture (only insertion of acupuncture needles into bilateral EX-B2, but without electrical stimulation) was applied to bilateral EX-B2 for 20 min, once daily, 6 times a week for 4 weeks. The hindlimb locomotor function (locomotor score) was assessed by using Faden's and colleagues' methods. The general conditions of rabbits in each group were observed, and their body weight changes were measured every week. Nucleus pulposus cells were isolated using enzyme digestion method. After the treatment, the Cav-1 positive cell counts in nucleus pulposus cells and annulus fibrosus tissues were detected by immunohistochemistry, and the telomerase activity of nucleus pulposus cells was detected by PCR-ELISA. The relative telomere length of nucleus pulposus cells was measured by real-time quantitative polymerase chain reaction (real-time qPCR), and the cell cycle of nucleus pulposus was detected by flow cytometry. RESULTS: Compared with the sham operation group, the body mass from 4 to 11 week, locomotor score at 4, 7 and 11 week, telomerase activity, relative telomere length and the proportion of cells in G2/M phase of nucleus pulposus cells were significantly decreased (P<0.01), while Cav-1 positive cell counts of nucleus pulposus and annulus fibrosus tissue, and the proportion of nucleus pulposus cells in the G0/G1 phase considerably increased (P<0.01) in the model group. Relevant to the model group, the EA group rather than the acupuncture group had an increase in the body mass from 8 to 11 week, locomotor score at 11 week, telomerase activity, relative telomere length of nucleus pulposus cells, and the proportion of nucleus pulposus cells in G2/M phase (P<0.01), and a decrease in the Cav-1 positive cell counts of nucleus pulposus and annulus fibrosus tissue and the proportion of cells in G0/G1 phase (P<0.01). No significant differences were found between the model and acupuncture groups in all the indexes mentioned above. CONCLUSIONS: EA at EX-B2 has a bene-ficial effect in improving motor function in rabbits with IVDD, which may be related to its functions in reducing the expression of Cav-1 in nucleus pulposus cells and annulus fibrosus, improving cycle arrest, enhancing the telomerase activity and the relative telomere length of nucleus pulposus cells, delaying the senescence of nucleus pulposus cells of the degenerated lumbar intervertebral discs.

Liang-Ge-San inhibits dengue virus serotype 2 infection by reducing caveolin1-induced cytoplasmic heat shock protein 70 translocation into the plasma membrane.

BACKGROUND: Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet. PURPOSE: In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored. METHODS: High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n = 10) and AG129 mice (n = 8) were used to examine the protective effects of LGS. RESULTS: It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads. CONCLUSION: This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.

Buyang Huanwu Decoction alleviates cerebral ischemic injury through modulating caveolin-1-mediated mitochondrial quality control.

Introduction: Mitochondrial quality control (MQC) is an important mechanism of neural repair after cerebral ischemia (CI). Recent studies have shown that caveolin-1 (Cav-1) is an important signaling molecule in the process of CI injury, but its mechanism of regulating MQC after CI is still unclear. Buyang Huanwu Decoction (BHD) is a classic traditional Chinese medicine formula that is often used to treat CI. Unfortunately, its mechanism of action is still obscure. Methods: In this study, we tested the hypothesis that BHD can regulate MQC through Cav-1 and exert an anti-cerebral ischemia injury effect. We used Cav-1 knockout mice and their homologous wild-type mice, replicated middle cerebral artery occlusion (MCAO) model and BHD intervention. Neurobehavioral scores and pathological detection were used to evaluate neurological function and neuron damage, transmission electron microscopy and enzymology detection of mitochondrial damage. Finally, western blot and RT-qPCR expression of MQC-related molecules were tested. Results: After CI, mice showed neurologic impairment, neuronal damage, and significant destruction of mitochondrial morphology and function, and MQC was imbalanced. Cav-1 deletion aggravated the damage to neurological function, neurons, mitochondrial morphology and mitochondrial function after CI, aggravated the imbalance of mitochondrial dynamics, and inhibited mitophagy and biosynthesis. BHD can maintain MQC homeostasis after CI through Cav-1 and improve CI injury. Discussion: Cav-1 can affect CI injury by regulating MQC, and this mechanism may be another target of BHD for anti-cerebral ischemia injury.

Emergency Treatment and Photoacoustic Assessment of Spinal Cord Injury Using Reversible Dual-Signal Transform-Based Selenium Antioxidant.

Spinal cord injury (SCI), following explosive oxidative stress, causes an abrupt and irreversible pathological deterioration of the central nervous system. Thus, preventing secondary injuries caused by reactive oxygen species (ROS), as well as monitoring and assessing the recovery from SCI are critical for the emergency treatment of SCI. Herein, an emergency treatment strategy is developed for SCI based on the selenium (Se) matrix antioxidant system to effectively inhibit oxidative stress-induced damage and simultaneously real-time evaluate the severity of SCI using a reversible dual-photoacoustic signal (680 and 750 nm). Within the emergency treatment and photoacoustic severity assessment (ETPSA) strategy, the designed Se loaded boron dipyrromethene dye with a double hydroxyl group (Se@BDP-DOH) is simultaneously used as a sensitive reporter group and an excellent antioxidant for effectively eliminating explosive oxidative stress. Se@BDP-DOH is found to promote the recovery of both spinal cord tissue and locomotor function in mice with SCI. Furthermore, ETPSA strategy synergistically enhanced ROS consumption via the caveolin 1 (Cav 1)-related pathways, as confirmed upon treatment with Cav 1 siRNA. Therefore, the ETPSA strategy is a potential tool for improving emergency treatment and photoacoustic assessment of SCI.

Polysaccharides from Panax ginseng promote intestinal epithelial cell migration through affecting the Ca2+ related regulators.

Background and aim: Panax ginseng, a key herbal medicine of replenishing Qi and tonifying Spleen, is widely used in the treatment of gastrointestinal diseases in East Asia. In this study, we aim to investigate the potential effects and mechanisms of polysaccharides from P. ginseng (PGP) on intestinal mucosal restitution which is one of the crucial repair modalities during the recovery of mucosal injury controlled by the Ca2+ signaling. Methods: Rat model of intestinal mucosal injury was induced by indomethacin. The fractional cell migration was carried out by immunohistochemistry staining with BrdU. The morphological observations on intestinal mucosal injury were also performed. Intestinal epithelial cell (IEC-6) migration in vitro was conducted by scratch method. Western-blot was adopted to determine the expressions of PLC-γ1, Rac1, TRPC1, RhoA and Cav-1. Immunoprecipitation was used to evaluate the levels of Rac1/PLC-γ1, RhoA/TRPC1 and Cav-1/TRPC1. Results: The results showed that PGP effectively reduced the assessment of intestinal mucosal injury, reversed the inhibition of epithelial cell migration induced by Indomethacin, and increased the level of Ca2+ in intestinal mucosa in vivo. Moreover, PGP dramatically promoted IEC-6 cell migration, the expression of Ca2+ regulators (PLC-γ1, Rac1, TRPC1, Cav-1 and RhoA) as well as protein complexes (Rac1/PLC-γ1, Cav-1/TRPC1 and RhoA/TRPC1) in vitro. Conclusion: PGP increases the Ca2+ content in intestinal mucosa partly through controlling the regulators of Ca2+ mobilization, subsequently promotes intestinal epithelial cell migration, and then prevents intestinal mucosal injury induced by indomethacin.

Biochemical and Biophysical Characterization of the Caveolin-2 Interaction with Membranes and Analysis of the Protein Structural Alteration by the Presence of Cholesterol.

Caveolin-2 is a protein suitable for the study of interactions of caveolins with other proteins and lipids present in caveolar lipid rafts. Caveolin-2 has a lower tendency to associate with high molecular weight oligomers than caveolin-1, facilitating the study of its structural modulation upon association with other proteins or lipids. In this paper, we have successfully expressed and purified recombinant human caveolin-2 using E. coli. The structural changes of caveolin-2 upon interaction with a lipid bilayer of liposomes were characterized using bioinformatic prediction models, circular dichroism, differential scanning calorimetry, and fluorescence techniques. Our data support that caveolin-2 binds and alters cholesterol-rich domains in the membranes through a CARC domain, a type of cholesterol-interacting domain in its sequence. The far UV-CD spectra support that the purified protein keeps its folding properties but undergoes a change in its secondary structure in the presence of lipids that correlates with the acquisition of a more stable conformation, as shown by differential scanning calorimetry experiments. Fluorescence experiments using egg yolk lecithin large unilamellar vesicles loaded with 1,6-diphenylhexatriene confirmed that caveolin-2 adsorbs to the membrane but only penetrates the core of the phospholipid bilayer if vesicles are supplemented with 30% of cholesterol. Our study sheds light on the caveolin-2 interaction with lipids. In addition, we propose that purified recombinant caveolin-2 can provide a new tool to study protein-lipid interactions within caveolae.

Tiao-bu-fei-shen formula promotes downregulation of the caveolin 1-p38 mapk signaling pathway in COPD - Associated tracheobronchomalacia cell model.

ETHNOPHARMACOLOGICAL RELEVANCE: The Tiao-bu-fei-shen (TBFS) formula, extensively used in Traditional Chinese Medicine (TCM), can enhance therapeutic efficacy and reduce the frequency of acute exacerbations of lung-kidney Qi deficiency in patients with chronic obstructive pulmonary disease (COPD). According to both TCM theory and long-term observation of practice, TBFS has become an effective treatment for COPD-associated tracheobronchomalacia (TBM). AIM OF THE STUDY: To investigate the mechanism of the TBFS formula in treating COPD-associated TBM based on caveolin 1-p38 MAPK signaling and apoptosis. MATERIALS AND METHODS: A rat COPD model was prepared by exposure to smoking combined with tracheal lipopolysaccharide injection. The trachea or bronchus chondrocytes from COPD rats were isolated, cultured, and treated with 10 ng/mL IL-1ß for 24 h to develop a model of COPD-associated TBM. Normal rats were administered TBFS to prepare drug-containing serum, and CCK8 assays were used to screen the optimal drug-containing serum concentration and SB203580 dose. TBFS drug-containing serum and SB203580 were processed separately for the control, model, drug-containing serum, blocker, and drug-containing serum combined with blocker groups. Flow cytometry and CCK8 assays were used to detect apoptosis and proliferative activity. Toluidine blue staining and immunohistochemistry were used to analyze the chondrocyte proteoglycan and type II collagen content. Western blotting was used to detect the expression of caveolin 1, p-p38 MAPK, TNF-α, IL-1ß, MMP-13, Bax, and Bcl-2 proteins. Quantitative PCR was used to detect the expression of caveolin 1, p38 MAPK, IL-1ß, MMP-13, Bax, Bcl-2, and miR-140-5p. RESULTS: The isolation and identification of bronchial chondrocytes from COPD rats revealed that 10 ng/mL IL-1ß can produce a stable COPD-associated TBM model. Screened via the CCK8 method, fourth-generation bronchial chondrocytes were determined as the optimal cells, and 5 µM SB203580 and 5% low-dose drug-containing serum were the optimal intervention doses. The experimental chondrocytes of each group were treated separately for 48 h. Toluidine blue staining and immunohistochemical analysis revealed that TBFS drug-containing serum, SB203580, and TBFS drug-containing serum combined with SB203580 can effectively increase the proteoglycan and type II collagen content after chondrocyte degradation. Flow cytometry of cells treated with SB203580 and TBFS drug-containing serum combined with SB203580 revealed significantly reduced cell apoptosis and enhanced cell proliferation activity. Western blot and qPCR analyses revealed that the TBFS drug-containing serum, SB203580, and TBFS drug-containing serum combined with SB203580 effectively inhibit the expression of caveolin 1, p-p38 MAPK, MMP-13, IL-1ß, TNF-α, and Bax proteins while promoting Bcl -2 protein expression. Treatment with TBFS drug-containing serum and SB203580 effectively inhibited the expression of MMP-13, p38 MAPK, caveolin 1, and Bax genes, and promoted the expression of Bcl-2 and miR-140-5p genes. CONCLUSIONS: A concentration of 10 ng/mL of IL-1ß can generate a stable COPD-associated TBM cell model. TBFS can improve the proteoglycan and type II collagen content, increase cell activity, and reduce the amount of chondrocyte apoptosis. The role of TBFS may be related to mechanisms of inhibiting the expression of the key signaling molecules caveolin 1 and p-p38 MAPK in the caveolin 1-p38 MAPK signaling pathway, thereby reducing the expression of the downstream effector products MMP-13, IL-1ß, and TNF-α, while inhibiting the expression of the apoptotic gene Bax and improving the expression of Bcl-2 and miR-140-5p genes.

Ursolic Acid Inhibits Breast Cancer Metastasis by Suppressing Glycolytic Metabolism via Activating SP1/Caveolin-1 Signaling.

Breast cancer remains the most common malignancy and the leading causality of cancer-associated mortality among women worldwide. With proven efficacy, Oldenlandia diffusa has been extensively applied in breast cancer treatment in Traditional Chinese Medicine (TCM) for thousands of years. However, the bioactive compounds of Oldenlandia diffusa accounting for its anti-breast cancer activity and the underlying biological mechanisms remain to be uncovered. Herein, bioactivity-guided fractionation suggested ursolic acid as the strongest anti-breast cancer compound in Oldenlandia diffusa. Ursolic acid treatment dramatically suppressed the proliferation and promoted mitochondrial-mediated apoptosis in breast cancer cells while brought little cytotoxicities in nonmalignant mammary epithelial cells in vitro. Meanwhile, ursolic acid dramatically impaired both the glycolytic metabolism and mitochondrial respiration function of breast cancer cells. Further investigations demonstrated that ursolic acid may impair the glycolytic metabolism of breast cancer cells by activating Caveolin-1 (Cav-1) signaling, as Cav-1 knockdown could partially abrogate the suppressive effect of ursolic acid on that. Mechanistically, ursolic acid could activate SP1-mediated CAV1 transcription by promoting SP1 expression as well as its binding with CAV1 promoter region. More meaningfully, ursolic acid administration could dramatically suppress the growth and metastasis of breast cancer in both the zebrafish and mouse xenotransplantation models of breast cancer in vivo without any detectable hepatotoxicity, nephrotoxicity or hematotoxicity. This study not only provides preclinical evidence supporting the application of ursolic acid as a promising candidate drug for breast cancer treatment but also sheds novel light on Cav-1 as a druggable target for glycolytic modulation of breast cancer.

Targeting the HIF-1α/Cav-1 Pathway with a Chicory Extract/Daidzein Combination Plays a Potential Role in Retarding Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most rapidly growing solid cancers, that is characterized by hypoxia. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates tumor proliferation and metastasis. It induces caveolin-1 (Cav-1) expression, a glycoprotein found on the membrane surface, then Cav-1 triggers angiogenesis and metastasis in HCC. OBJECTIVE: We hypothesize that targeting HIF-1α and consequently, Cav-1 using the antioxidant natural compound such as chicoric acid and a Cav-1 inhibitor daidzein (DAZ) could be a useful approach in the management of HCC. This study was conducted to investigate the possible therapeutic efficacy of standardized chicory leaf extract (SCLE) and DAZ via modulation of HIF-1α and Cav-1 in HCC rats. METHODS: Diethyl nitrosamine (DENA) was used for HCC induction. After the induction period, four groups (10 rats for each) were treated with SCLE, DAZ, a combination of both, as well as sorafenib, all compared to the non-treated control. We assessed hepatic HIF-1α protein expression, Cav-1 gene expression, serum level of AFP, hepatic tissue content of VEGF, MMP-9, oxidative stress markers MDA and SOD. RESULTS: DAZ, SCLE, and their combination, significantly down-regulated the expression of HIF-1α, Cav-1, and consequently dampened MMP-9, VEGF, hepatic content. It has been observed that the combination treatment showed a synergistic effect compared to either treatment alone. Importantly, the combination treatment exhibited a significantly more potent effect than sorafenib. CONCLUSION: This study showed the potential role of the HIF-1α/Cav-1 pathway in HCC progression, moreover, SCLE and DAZ showed a potent efficacy in retarding HCC via modulation of this pathway.

Notoginsenoside R1 intervenes degradation and redistribution of tight junctions to ameliorate blood-brain barrier permeability by Caveolin-1/MMP2/9 pathway after acute ischemic stroke.

BACKGROUND: The leakage of blood-brain barrier (BBB) is main pathophysiological change in acute stage of ischemic stroke, which not only deteriorates neurological function, but also increases the risk of hemorrhagic transformation after thrombolysis. PURPOSE/STUDY DESIGN: This article investigates the efficacy of Notoginsenoside R1, an active ingredient of Panax notoginseng, on BBB permeability and explores related mechanisms after acute ischemic stroke. METHODS: In vivo, male Sprague-Dawley rats (260-280 g) were selected and randomly divided into 6 groups: sham group, model group, low, middle and high doses of Notoginsenoside R1 groups and positive drug Dl-3-n-Butylphthalide group. Except for sham group, rats were performed with permanent middle cerebral artery occlusion model in each group. Twelve hours later, rats were evaluated for Bederson neurological function, and BBB integrity by Evans blue leak imaging; Triphenyltetrazolium chloride staining was used to detect the volume of cerebral infarction. Frozen sections of rats' brain tissue were prepared for detection of MMPs activity in situ zymography. Peripheral tissue of cerebral infarction was collected and tested the expression of MMP2, 9 and tight junction proteins (zo1, claudin5, occludin) by western blot. In vitro, transwell endothelial barrier model was established by bEnd.3 cells. Oxygen glucose deprivation (OGD) was chosen to simulate the hypoxic environment. Suitable OGD stimulation time as well as Notoginsenoside R1 and Dl-3-n-Butylphthalide optimal dose concentrations were determined through transwell leakage and CCK8 assay. Furthermore, endothelial subcellular component proteins were extracted. The change of zo1, claudin5, occludin and caveolin1 was detected by western blot. RESULTS: Notoginsenoside R1 treatment significantly reduced BBB leakage and cerebral infarction volume, weakened neurological deficits in post-stroke rats. Moreover, it inhibited the activity of MMPs in infarcted cortex and striatum, down-regulated MMP2, 9 and up-regulated zo1 and claudin5 expressions in penumbra. In vitro, Notoginsenoside R1 treatment decreased OGD-induced endothelial barrier permeability, restored expressions of zo1, claudin5 on cellular membrane and cytoplasm, as well as mediated membrane redistribution of occludin and caveolin1 from actin cytoskeletal fraction. CONCLUSIONS: Notoginsenoside R1 treatment attenuates BBB permeability, cerebral infarction volume and neurological impairments in rats with acute cerebral ischemia. The mechanisms might be related to intervening degradation and redistribution of zo1, caludin5 and occludin by caveolin1/ MMP2/9 pathway. More effects and mechanisms of Notoginsenoside R1 on rehabilitation of stroke are worthy to be explored in the future.

Recent developments in the regulation of cholesterol transport by natural molecules.

The dysregulation of cholesterol metabolism is a high-risk factor for non-alcoholic fatty liver disease (NAFLD), dyslipidemia, and atherosclerosis (AS). Cholesterol transport maintains whole-body cholesterol homeostasis. Low-density apolipoprotein receptor (LDLR) mediates cholesterol uptake in cells and plays an important role in the primary route of circulatory cholesterol clearance in liver cells. Caveolins 1 is an integral membrane protein and shuttle between the cytoplasm and cell membrane. Caveolins 1 not only plays a role in promoting cholesterol absorption in cells but also in the transport of cellular cholesterol efflux by interacting with the ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI). These proteins, which are associated with reverse cholesterol transport (RCT), are potential therapeutic targets for NAFLD and AS. Many studies have indicated that natural products have lipid-lowering effects. Moreover, natural molecules, derived from natural products, have the potential to be developed into novel drugs. However, the mechanisms underlying the regulation of cholesterol transport by natural molecules have not yet been adequately investigated. In this review, we briefly describe the process of cholesterol transport and summarize the mechanisms by which molecules regulate cholesterol transport. This article provides an overview of recent studies and focuses on the potential therapeutic effects of natural molecules; however, further high-quality studies are needed to firmly establish the clinical efficacies of natural molecules.

Autophagic Inhibition of Caveolin-1 by Compound Phyllanthus urinaria L. Activates Ubiquitination and Proteasome Degradation of ß-catenin to Suppress Metastasis of Hepatitis B-Associated Hepatocellular Carcinoma.

Compound Phyllanthus urinaria L. (CP) is a traditional Chinese medicine (TCM) formula for cancer treatment in the clinic, particularly during progression of hepatitis B-associated hepatocellular carcinoma (HBV-associated HCC). Nevertheless, its anti-metastatic action and mechanisms are not well elucidated. In this study, CP was found to exert remarkable inhibitory effects on the proliferation, migration and invasion of HBV-associated HCC cells. The following network and biological analyses predicted that CP mainly targeted Caveolin-1 (Cav-1) to induce anti-metastatic effects, and Wnt/ß-catenin pathway was one of the core mechanisms of CP action against HBV-associated HCC. Further experimental validation implied that Cav-1 overexpression promoted metastasis of HBV-associated HCC by stabilizing ß-catenin, while CP administration induced autophagic degradation of Cav-1, activated the Akt/GSK3ß-mediated proteasome degradation of ß-catenin via ubiquitination activation, and subsequently attenuated the metastasis-promoting effect of Cav-1. In addition, the anti-cancer and anti-metastatic action of CP was further confirmed by in vivo and ex vivo experiments. It was found that CP inhibited the tumor growth and metastasis of HBV-associated HCC in both mice liver cancer xenograft and zebrafish xenotransplantation models. Taken together, our study not only highlights the novel function of CP formula in suppressing metastasis of HBV-associated HCC, but it also addresses the critical role of Cav-1 in mediating Akt/GSK3ß/ß-catenin axis to control the late-phase of cancer progression.

Decrease of ABCB1 protein expression and increase of G1 phase arrest induced by oleanolic acid in human multidrug-resistant cancer cells.

Oleanolic acid (OA) is a natural compound that can be found in a number of edible and medicinal plants and confers diverse biological actions. However, the direct target of OA in human tumor cells remains poorly understood, preventing its application in clinical and health settings. A previous study revealed that overexpression of caveolin-1 in human leukemia HL-60 cells can increase its sensitivity to OA. The present study aimed to investigate the effects of OA on the doxorubicin-resistant human breast cancer MCF-7 cell line (MCF-7/DOX), harringtonine-resistant human leukemia HL-60 cells (HL-60/HAR) and their corresponding parental cell lines. Western blotting was performed to measure protein expression levels, whilst Cell Counting Kit-8 (CCK-8) assays, cell cycle analysis (by flow cytometry) and apoptosis assays (with Annexin V/PI staining) were used to assess drug sensitivity. CCK-8 assay results suggested that MCF-7/DOX cells, which overexpress the caveolin-1 protein, have similar OA susceptibility to their parent line. In addition, sensitivity of MCF-7/DOX cells to OA was not augmented by knocking down caveolin-1 using RNA interference. HL-60/HAR cells exhibited a four-fold increased sensitivity to OA compared with that in their parental HL-60 cells according to CCK-8 assay. Both of the resistant cell lines exhibited higher numbers of cells at G1 phase arrest compared with those in their parent lines, as measured via flow cytometry. Treatment of both MCF-7 cell lines with 100 µM OA for 48 h induced apoptosis, with increased effects observed in resistant cells. However, no PARP-1 or caspase-3 cleavage was observed, with some positive Annexin V staining found after HL-60/HAR cells were treated with OA, suggesting that cell death occurred via non-classical apoptosis or through other cell death pathways. It was found that OA was not a substrate of ATP-binding cassette subfamily B member 1 (ABCB1) in drug-resistant cells, as indicated by the accumulation of rhodamine 123 assessed using flow cytometry. However, protein expression of ABCB1 in both of the resistant cell lines was significantly decreased after treatment with OA in a concentration-dependent manner. Collectively, these results suggest that OA could reduce ABCB1 protein expression and induce G1 phase arrest in multidrug-resistant cancer cells. These findings highlight the potential of OA for cancer therapy.

Oxymatrine improves blood-brain barrier integrity after cerebral ischemia-reperfusion injury by downregulating CAV1 and MMP9 expression.

BACKGROUND: Ischemic stroke (IS) is a major neurological condition associated with extremely high morbidity and mortality worldwide. Oxymatrine (OMT), a quinolizidine alkaloid extracted from the root of Sophora flavescens, has neuroprotective properties and protects against IS. However, whether its protective effect involves alterations in the integrity of the blood-brain barrier (BBB) is unknown. PURPOSE: Here, we used in vivo and in vitro models of IS to evaluate the protective effects of OMT and to establish whether its effects are mediated via the modulation of the BBB function. METHODS: We assessed the effects of OMT by using neurological function scores, triphenyltetrazolium chloride staining, Nissl staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling. RESULTS: OMT significantly prevented cellular damage, improved neurological function, and reduced BBB permeability in a mouse model of cerebral ischemia-reperfusion. Additionally, OMT protected the function of the tight junctions of bEend.3 cells against the consequences of oxygen-glucose deprivation. Furthermore, intracranial lentivirus injection of short hairpin RNA targeting Cav1 decreased caveolin-1 expression and inhibited the neuroprotective effects of OMT. CONCLUSIONS: OMT attenuated ischemia-reperfusion injury-induced damage to the BBB, and this neuroprotective action was at least partially dependent on the expression levels of CAV1 and MMP9 proteins. Therefore, OMT may offer effective protection against BBB injury induced by ischemia-reperfusion episodes.

Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function.

Caveolin-1 (Cav-1) is a scaffolding protein and a major component of caveolae/lipid rafts. Previous reports have shown that endothelial dysfunction in Cav-1-deficient (Cav-1-/-) mice is mediated by elevated oxidative stress through endothelial nitric oxide synthase (eNOS) uncoupling and increased NADPH oxidase. Oxidant stress is the net balance of oxidant generation and scavenging, and the role of Cav-1 as a regulator of antioxidant enzymes in vascular tissue is poorly understood. Extracellular SOD (SOD3) is a copper (Cu)-containing enzyme that is secreted from vascular smooth muscle cells/fibroblasts and subsequently binds to the endothelial cells surface, where it scavenges extracellular [Formula: see text] and preserves endothelial function. SOD3 activity is dependent on Cu, supplied by the Cu transporter ATP7A, but whether Cav-1 regulates the ATP7A-SOD3 axis and its role in oxidative stress-mediated vascular dysfunction has not been studied. Here we show that the activity of SOD3, but not SOD1, was significantly decreased in Cav-1-/- vessels, which was rescued by re-expression of Cav-1 or Cu supplementation. Loss of Cav-1 reduced ATP7A protein, but not mRNA, and this was mediated by ubiquitination of ATP7A and proteasomal degradation. ATP7A bound to Cav-1 and was colocalized with SOD3 in caveolae/lipid rafts or perinucleus in vascular tissues or cells. Impaired endothelium-dependent vasorelaxation in Cav-1-/- mice was rescued by gene transfer of SOD3 or by ATP7A-overexpressing transgenic mice. These data reveal an unexpected role of Cav-1 in stabilizing ATP7A protein expression by preventing its ubiquitination and proteasomal degradation, thereby increasing SOD3 activity, which in turn protects against vascular oxidative stress-mediated endothelial dysfunction.

[Inhibitory effect of paeonol on aortic endothelial inflammation in atherosclerotic rats by up-regulation of caveolin-1 expression and suppression of NF-κB pathway].

To explore whether paeonol can play an anti-atherosclerotic role by regulating the expression of aortic caveolin-1 and affecting NF-κB pathway, so as to inhibit the inflammatory response of vascular endothelium in atherosclerotic rats. The atherosclerotic model of rats was induced by high-fat diet and vitamin D_2. The primary culture of vascular endothelial cells(VECs) was carried out by tissue block pre-digestion and adherent method. The injury model of VECs was induced by lipopolysaccharide(LPS), and filipin, a small concave protein inhibitor, was added for control. HE staining was used to observe pathological changes of aorta. TNF-α, IL-6 and VCAM-1 were detected by ELISA. Western blot assay was used to detect the protein expression levels of caveolin-1 and p65 in aorta and VECs. The results showed that as compared with model group, paeonol significantly reduced aortic plaque area and lesion degree in rats, decreased the level of serum TNF-α, IL-6 and VCAM-1 in the rats and enhanced the relative expression level of caveolin-1, decreased p65 expression conversely(P<0.05 or P<0.01). In vitro, as compared to model group, paeonol obviously improved cell morphology, decreased the secretion of TNF-α, IL-6 and VCAM-1 in VECs, increased caveolin-1 expression, and decreased p65 protein expression(P<0.05 or P<0.01). Furthermore, filipin could reverse the effect of paeonol on expression of inflammatory factors and proteins(P<0.05 or P<0.01). According to the results, it was found that paeonol could play the role of anti-atherosclerosis by up-regulating the expression of caveolin-1 and inhibiting the activation of NF-κB pathway to reduce vascular inflammation in atherosclerotic rats.

Multifaceted Functions of Host Cell Caveolae/Caveolin-1 in Virus Infections.

Virus infection has drawn extensive attention since it causes serious or even deadly diseases, consequently inducing a series of social and public health problems. Caveolin-1 is the most important structural protein of caveolae, a membrane invagination widely known for its role in endocytosis and subsequent cytoplasmic transportation. Caveolae/caveolin-1 is tightly associated with a wide range of biological processes, including cholesterol homeostasis, cell mechano-sensing, tumorigenesis, and signal transduction. Intriguingly, the versatile roles of caveolae/caveolin-1 in virus infections have increasingly been appreciated. Over the past few decades, more and more viruses have been identified to invade host cells via caveolae-mediated endocytosis, although other known pathways have been explored. The subsequent post-entry events, including trafficking, replication, assembly, and egress of a large number of viruses, are caveolae/caveolin-1-dependent. Deprivation of caveolae/caveolin-1 by drug application or gene editing leads to abnormalities in viral uptake, viral protein expression, or virion release, whereas the underlying mechanisms remain elusive and must be explored holistically to provide potential novel antiviral targets and strategies. This review recapitulates our current knowledge on how caveolae/caveolin-1 functions in every step of the viral infection cycle and various relevant signaling pathways, hoping to provide a new perspective for future viral cell biology research.