Lycorine inhibits angiogenesis by docking to PDGFRα.

Lycorine (Lyc) is a natural alkaloid derived from medicinal plants of the Amaryllidaceae family. Lyc has been reported to inhibit the recurrence and metastasis of different kinds of tumors. However, Lyc's effect on angiogenesis and its specific mechanism are still not clear. This study was designed to test the antiangiogenesis effect of Lyc and to explore the possible mechanisms. We performed cell experiments to confirm Lyc's inhibitory effect on angiogenesis and employed sunitinib as a positive control. Moreover, the synergistic effect of Lyc and sunitinib was also explored. Next, we conducted bioinformatics analyses to predict the potential targets of Lyc and verified them by western blotting and immunofluorescence. Molecular docking, kinase activity assays, Biacore assays and cellular thermal shift assays (CETSAs) were applied to elucidate the mechanism by which Lyc inhibited target activity. Lyc inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs). Employing bioinformatics, we found that Lyc's target was PDGFRα and that Lyc attenuated PDGFRα phosphorylation. We also found that Lyc inhibited PDGFRα activation by docking to it to restrain its activity. Additionally, Lyc significantly inhibited PDGF-AA-induced angiogenesis. This study provides new insights into the molecular functions of Lyc and indicates its potential as a therapeutic agent for tumor angiogenesis.

An extraction from Trametes robiniophila Murr. (Huaier) inhibits non-small cell lung cancer proliferation via targeting to epidermal growth factor receptor.

An extraction from Trametes robiniophila Murr. (Huaier) is a kind of natural fungus growing from the sophora japonica tree. Huaier is widely applied to cure the hepatocellular cancer (HCC). However, the medicinal fungus' curative result on non-small-cell lung cancer (NSCLC) is not well elaborated. In this study, we applied in vitro experiments to study Huaier's curative result on NSCLC. The potential Huaier targets were predicted using bioinformatics and validated by western blotting. We further elucidated the mechanism of Huaier targeting by molecular docking, kinase activity assay, CEllular Thermal Shift Assays (CETSAs). At last, in vivo curative result was verified further. Huaier weakened proliferation and promoted apoptosis of the NSCLC cell lines. According to bioinformatics, Epidermal Growth Factor Receptor (EGFR) may be the target of Huaier. Western blotting showed that Huaier can attenuate the activation of EGFR and we found that Huaier can dock to EGFR. Huaier significantly inhibited the tumor growth by weakening the expression of p-EGFR in vivo. This study offers a new idea for further understanding of Huaier and shows its potential as a therapeutic agent.

Bufalin inhibits human diffuse large B-cell lymphoma tumorigenesis by inducing cell death through the Ca2+/NFATC1/cMYC pathway.

The 5-year survival rate of diffuse large B-cell lymphoma (DLBCL) can reach 60%. However, nearly half of patients undergo relapse/refractory issues with a survival period of less than 2 years. New therapeutic approaches are therefore needed to improve chemotherapy efficacy and patient survival. Bufalin (BF), isolated from the traditional Chinese medicine Chansu, has been reported to play an anticancer role in multiple cancer cell types. However, there are few reports of the effects of BF on the growth of DLBCL. In the present study, we demonstrated that BF exerts antitumor activity in DLBCL cells, both in vitro and in vivo. Treatment of DLBCL cells with BF resulted in increased proliferation and apoptosis in a dose- and time-dependent manner. Daily intraperitoneal injection of 1.5 mg/kg BF significantly delayed DLBCL xenograft growth in NOD/SCID mice without affecting body weight. Bioinformatics analysis showed that BF may regulate NFATC1 protein and affect expression of its downstream gene, cMYC. Our results suggest that BF can attenuate NFATC1 translocation by reducing the intracellular calcium concentration; BF may also have a low synergistic effect with cyclosporin A. In conclusion, we demonstrated that BF exerts antitumor activity that is mediated at least in part by the Ca2+/NFATC1/cMYC pathway. Our findings suggest that BF can be effectively applied as a novel potential therapeutic agent for DLBCL.

Piperlongumine inhibits head and neck squamous cell carcinoma proliferation by docking to Akt.

Piperlongumine (PL) is a biologically active alkaloid isolated from the long pepper roots and widely used as a traditional medicine in Ayurvedic medicine. However, the mechanism of PL's effect on head and neck squamous cell carcinoma (HNSCC) is not well understood. We performed cell experiments to confirm PL's inhibitory effect on HNSCC and employing cisplatin as positive control. Next, we conducted bioinformatics to predict PL's potential targets and verified by western blotting. Molecular docking, Biacore experiment and kinase activity assays were applied to elucidate the mechanism by which PL inhibited target activity. In vivo efficacy was verified by xenotransplantation and immunohistochemistry. PL inhibited proliferation, promoted late apoptosis, arrested cell cycle and inhibited DNA replication of the HEp-2 and FaDu cell lines. Employing bioinformatics, we found that PL's target was Akt and PL attenuated Akt phosphorylation. We found from molecular docking, Biacore experiment and kinase activity assay that PL inhibited Akt activation by docking to Akt to restrain its activity. In addition, PL significantly inhibited the growth of xenograft tumors by down regulating the expression of p-Akt in vivo. This study provides new insights into the molecular functions of PL and indicate its potential as a therapeutic agent for HNSCC.

ß-Elemene inhibits the metastasis of multidrug-resistant gastric cancer cells through miR-1323/Cbl-b/EGFR pathway.

BACKGROUND: ß-Elemene is a natural agent extracted from the traditional Chinese herbal medicine Curcuma wenyujin that is a promising novel plant-derived drug with broad-spectrum anticancer activity. Our previous study identified an enhanced capacity for metastasis in multidrug resistant (MDR) gastric cancer and breast cancer cells. However, the anti-metastatic effects of ß-Elemene on MDR cancer cells remain unknown. PURPOSE: In this study, we posit the hypothesis that ß-elemene possesses antimetastatic effects on MDR cancer cells. METHODS: Cell viability assay was used to assess the resistance of SGC7901/ADR cells and the cytotoxic effects of ß-Elemene. Wound healing, transwell assay and lung metastatic mice model were used to the anti-metastasis effects of ß-Elemene. MicroRNA microarray analysis was used to explore potential regulated miRNAs. Luciferase reporter assay was used to identify the direct target. Human MMP antibody array, western blot, immunoprecipitation, qRT-PCR analyses and immunohistochemistry were conducted to investigate the underlying anti-metastasis mechanism of ß-Elemene. RESULTS: In this study, we found that ß-Elemene significantly inhibited the metastatic capacity of MDR gastric cells in vivo and in vitro. Mechanistically, we found that ß-Elemene regulated MMP-2/9 expression and reversed epithelial-mesenchymal transition. Further studies showed that ß-Elemene upregulated Cbl-b expression, resulting in inhibition of the EGFR-ERK/AKT pathways, which regulate MMP-2/9. Additionally, we confirmed that ß-Elemene upregulated Cbl-b by inhibiting miR-1323 expression. Finally, we found that numbers of metastatic tumor nodules were significantly decreased in the lungs of nude mice after ß-Elemene treatment. CONCLUSION: Our results suggested that ß-Elemene inhibits the metastasis of MDR gastric cancer cells by modulating the miR-1323/Cbl-b/EGFR signaling axis.

Effects of Baihui electroacupuncture in a rat model of depression.

BACKGROUND: This study aimed to investigate the effect of electroacupuncture (EA) on behavior in a rat model of chronic unpredictable mild stress (CUMS) and to explore the underlying molecular mechanisms. METHODS: A total of 45 adult male Sprague-Dawley rats were randomly divided into three groups: the control, CUMS, and CUMS plus EA groups. Rats in the CUMS and EA groups were subjected to a 3-week CUMS condition, while rats in the EA group received EA at the Baihui (GV 20) acupoint (2 Hz, 0.6 mA) for 10 min once daily before being subjected to the CUMS condition. The sucrose preference test (SPT) was used as a measure to infer activation of the pleasure response to depression-like behaviour. After the behavioral test, 5-bromodeoxyuridine (BrdU) was intraperitoneally injected (100 mg/kg) and brain samples were collected 24 h later for the detection of hippocampal BrdU. Cell proliferation was determined according to the proportion of BrdU-positive cells. Brain-derived neurotrophic factor (BDNF) expression was detected. RESULTS: The severity of anhedonia, BDNF+ cells, and BrdU+ neurons in DG significantly decreased in CUMS rats, and was accompanied by a reduced BDNF and BrdU+ expression (P<0.05). After EA, the low levels of BDNF+ cells and BrdU+ expression and the depression-like behavior increased markedly (P<0.05). CONCLUSIONS: EA contributes to neuroprotection against CUMS by enhancing BDNF expression and improving hippocampal neurogenesis.

WRKY44 represses expression of the wound-induced sesquiterpene biosynthetic gene ASS1 in Aquilaria sinensis.

Agarwood is derived from wounds in Aquilaria trees and is widely used in traditional medicine, incense, and perfume. Sesquiterpenes are one of the main active components in agarwood and are known to be induced by wounding or injury; However, the molecular mechanisms by which wounding leads to sesquiterpene formation remain largely unknown. Agarwood sesquiterpene synthase 1 (ASS1) is one of key enzymes responsible for the biosynthesis of sesquiterpenes and is a crucial jasmonate (JA)-responsive wound-inducible synthase. However, it is not known why ASS1 is not expressed in healthy trees and how its expression is induced as a result of wounding. Here, we report that ASS1 is a wound-induced gene with a promoter in which a 242-bp region (-973 to -731bp) is identified as the core sequence for responding to wound signals. AsWRKY44 binds directly to this region and represses ASS1 promoter activity. Down-regulation or disruption of AsWRKY44 can relieve the inhibition and activate ASS1 expression. In addition, AsWRKY44 is degraded and the expression of ASS1 is significantly up-regulated in response to exogenous application of methyl jasmonate. Thus, AsWRKY44 is a crucial negative regulator of wound-induced ASS1 transcription, and is central to the mechanism of sesquiterpene biosynthesis in agarwood.

SNX17 (Sorting Nexin 17) Mediates Atrial Fibrillation Onset Through Endocytic Trafficking of the Kv1.5 (Potassium Voltage-Gated Channel Subfamily A Member 5) Channel.

BACKGROUND: Kv1.5 (Potassium voltage-gated channel subfamily A member 5) has been regarded as a promising target of interventions for atrial fibrillation (AF). SNX17 (sorting nexin 17), a member of the SNXs (sorting nexin family), regulates the intracellular trafficking of membrane proteins through its FERM (four-point-one, ezrin, radixin, moesin) domain. However, whether SNX17 regulates the trafficking process of Kv1.5 remains unknown. METHODS: A SNX17 knockout rat line was generated to test the role of SNX17 in atrial electrophysiology. The protein expression of SNX17 and membrane ion channels was detected by Western blotting. Electrophysiology changes in the atrial tissue and myocytes were analyzed by optical mapping and patch clamp, respectively. Acetylcholine and electrical stimulation were used to induce AF, and ECG recording was adopted to assess the influence of SNX17 deficiency on AF susceptibility. The spatial relationship between Kv1.5 and SNX17 was evaluated by immunostaining and confocal scanning, and the functional region of SNX17 regulating Kv1.5 trafficking was identified using plasmids with truncated SNX17 domains. RESULTS: Embryonic death occurred in homozygous SNX17 knockout rats. SNX17 heterozygous rats survived, and the level of the SNX17 protein in the atrium was decreased by ≈50%. SNX17 deficiency increased the membrane expression of Kv1.5 and atria-specific ultrarapid delayed rectifier outward potassium current ( IKur) density, resulting in a shortened action potential duration, and eventually contributing to AF susceptibility. Mechanistically, SNX17 facilitated the endocytic sorting of Kv1.5 from the plasma membrane to early endosomes via the FERM domain. CONCLUSIONS: SNX17 mediates susceptibility to AF by regulating endocytic sorting of the Kv1.5 channel through the FERM domain. SNX17 could be a potential target for the development of new drugs for AF.

Berberine ameliorates renal injury by regulating G proteins-AC- cAMP signaling in diabetic rats with nephropathy.

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to glomerulus and glomerular mesangial cells (MCs) proliferation play a critical role in the pathogenesis of DN. The current studies were undertaken to investigate the protective effects and the possible molecular mechanism of berberine on streptozotocin (STZ)-induced DN rats. Male Wistar rats were randomly assigned to normal control and DN groups of comparable age. Three DN groups received 50, 100 and 200 mg/kg of berberine for 8 weeks via daily intragastrically, respectively. The G proteins-adenylyl cyclase (AC)-cAMP signaling pathway and glomerular MCs proliferation were examined in STZ-induced diabetic rat kidney. Enhanced MCs proliferation and remarkable renal injury were concomitant with activation of Gαi and inhibition of Gαs and cAMP in DN model group. Berberine treatment for 8 weeks abolished the above changes by upregulating the expression of Gαs protein and downregulating the expression of Gαi protein, increasing cAMP level, and inhibiting MCs proliferation compared with model group. Taken together, for the first time, these results demonstrated that berberine can relieve renal injury in DN rats through mediating G proteins-AC-cAMP signaling pathway and inhibiting the abnormal proliferation of MCs by increasing cAMP level, suggesting that berberine could be a potential therapeutic agent for the treatment of DN.

Effects of cyclodextrins on the antimicrobial activity of plant-derived essential oil compounds.

Essential oils (EOs) from plants are considered to be a safer alternative when compared to synthetic antimicrobial food additives. However, a major drawback of many EOs is their hydrophobic nature, which makes them insoluble in water based media and matrices. Although cyclodextrins (CDs) can increase the solubility of EO compounds, the effects of CDs on the antimicrobial activity of EOs have not been reported. In this paper, four different EO compounds (carvacrol, eugenol, linalool and 2-pentanoylfuran) were chosen to study the influence of CDs on the solubility and antimicrobial activity on bacteria and yeast. The greatest enhancement with regards to solubility of the four test compounds was achieved by hydroxypropyl-ß-CD. In most instances, not only were the minimal antimicrobial concentrations of EO compounds decreased, but the interactivity of two combined EO compounds could be strengthened by the co-addition of CDs. Furthermore, the combination of carvacrol with hydroxypropyl-ß-CD caused a marked change in the major membrane lipid composition of all microorganisms investigated; while scanning electron microscopy revealed that cellular integrity was significantly affected by 2× MIC, ultimately resulting in cell lysis.

[Effect of berberine on expression of transforming growth factor-beta1 and type IV collagen proteins in mesangial cells of diabetic rats with nephropathy].

OBJECTIVE: To explore the effect and mechanism of berberine on diabetic nephropathy in experimental rats. METHOD: The rat model of diabetic nephropathy was induced by injection of streptozocin (STZ). The rats were divided into 6 groups: control group, model group, 3 berberine treatment groups and Xiaoke Wan (XKW) treatment group. The fasting blood glucose (FBG), kidney weight/body weight (KW/BW), blood urea nitrogen (BUN), creatinine (Cr) and urinary protein (Upro) were tested 8 weeks later. The expression of transforming growth factor-beta1 (TGF-beta1) and type IV collagen (IV-C) proteins in renal tissue of diabetic rats with nephropathy were observed by optical micrography. RESULT: Berberine could reduce the levels of FBG, KW/BW, BUN, Cr, Upro and the expression of TGF-beta1 and IV-C proteins in renal tissue of diabetic rats with nephropathy. CONCLUSION: Berberine may protect renal function and slow down the progression of diabetic nephropathy in rats by suppressing the expression of TGF-beta1 and IV-C proteins in renal tissue.

[The effects of safflower injection on lipid peroxidation level and expression of heme oxygenase-1 of the rat liver with hypoxia and hypercapnia].

AIM: To investigate the changes of lipid peroxidation level and expression of heme oxygenase-1 of the rat liver with chronic hypoxia and hypercapnia, and the effects of Safflower injection (a compond of Chinese Traditional medicine). METHODS: Thirty male SD rats weighing 180 approximately 220 g were divided into three groups (n=10): control group (N group), chronic hypoxia and hypercapnia for four weeks group(F group), and Safflower injection group (H group). SOD and MDA in liver tissue were measured by spectrophotometric method. And methods Immunohistochemical assay was used to detect the distribution of HO-1 protein. Pathological changes in liver tissues were observed in HE staining section. The mRNA expressions of HO-1 in liver were detected by semi-quantitative RT-PCR. RESULTS: The activity of SOD of the liver in F group were significantly lower than those in N group, and the content of MDA were significantly higher. The activity of SOD of the liver in H group were significantly higher than those in F group, and the content of MDA were significantly lower. In F group there were multiple dispersed immunoreactivity cells in liver. And compared to those in F group, the immunoreactivity cells were significantly decreased in H group. HE staining revealed that there were many hepatocytes with obvious adipose degeneration. Hepatic pathological damage in H group was slighter than that in F group. The expression of HO-1 mRNA of the liver in F group were significantly higher than those in N group (P < 0.01), and those in H group were significantly lower than those in F group (P < 0.01) . CONCLUSION: Chronic hypoxia and hypercapnia increases the level of oxidative stress. Safflower injection have a protective effect, maybe because of the accommodation of the expression of HO-1 of the liver and the elimination of free radicals.

The effects of safflower injection on lipid peroxidation level and expression of heme oxygenase-1 of the rat liver with hypoxia and hypercapnia / 中国应用生理学杂志

<p><b>AIM</b>To investigate the changes of lipid peroxidation level and expression of heme oxygenase-1 of the rat liver with chronic hypoxia and hypercapnia, and the effects of Safflower injection (a compond of Chinese Traditional medicine).</p><p><b>METHODS</b>Thirty male SD rats weighing 180 approximately 220 g were divided into three groups (n=10): control group (N group), chronic hypoxia and hypercapnia for four weeks group(F group), and Safflower injection group (H group). SOD and MDA in liver tissue were measured by spectrophotometric method. And methods Immunohistochemical assay was used to detect the distribution of HO-1 protein. Pathological changes in liver tissues were observed in HE staining section. The mRNA expressions of HO-1 in liver were detected by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>The activity of SOD of the liver in F group were significantly lower than those in N group, and the content of MDA were significantly higher. The activity of SOD of the liver in H group were significantly higher than those in F group, and the content of MDA were significantly lower. In F group there were multiple dispersed immunoreactivity cells in liver. And compared to those in F group, the immunoreactivity cells were significantly decreased in H group. HE staining revealed that there were many hepatocytes with obvious adipose degeneration. Hepatic pathological damage in H group was slighter than that in F group. The expression of HO-1 mRNA of the liver in F group were significantly higher than those in N group (P < 0.01), and those in H group were significantly lower than those in F group (P < 0.01) .</p><p><b>CONCLUSION</b>Chronic hypoxia and hypercapnia increases the level of oxidative stress. Safflower injection have a protective effect, maybe because of the accommodation of the expression of HO-1 of the liver and the elimination of free radicals.</p>

[Isolation and purification of total tubeimosides from Bolbostemma paniculatum by macroporous resin].

OBJECTIVE: To select the optimum purification process of all tubeimosides from Bolbostemma paniculatum by macroporous resin. METHOD: Static absorption test and deabsorption test were carried out to screen the best macroporous resin for all tubeimosides. The single factor test, orthogonal experimental designs and variance analysis were applied to optimize the manipulation parameters of macroporous resin. RESULT: The macroporous resin NKA possessed the strongest absorption ability to all tubeimosides among the 9 resins studied, in addition to an easy de-absorption property. The optimum absorption conditions were A2B3C1, namely concentration of feed 30 mg x mL(-1), diameter: height of the chromatography column 1:7, the weight ratio of raw material and resins 1:2. CONCLUSION: The above experimental results can provide experimental basis for large-scale purification process of all tubeimosides.