Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts.

AIM: To investigate the inhibitory effect of astragaloside IV on the pathological functions of cancer-associated fibroblasts, and to explore the underlying mechanism. METHODS: Paired gastric normal fibroblast (GNF) and gastric cancer-associated fibroblast (GCAF) cultures were established from resected tissues. GCAFs were treated with vehicle control or different concentrations of astragaloside IV. Conditioned media were prepared from GNFs, GCAFs, control-treated GCAFs, and astragaloside IV-treated GCAFs, and used to culture BGC-823 human gastric cancer cells. Proliferation, migration and invasion capacities of BGC-823 cells were determined by MTT, wound healing, and Transwell invasion assays, respectively. The action mechanism of astragaloside IV was investigated by detecting the expression of microRNAs and the expression and secretion of the oncogenic factor, macrophage colony-stimulating factor (M-CSF), and the tumor suppressive factor, tissue inhibitor of metalloproteinase 2 (TIMP2), in different groups of GCAFs. The expression of the oncogenic pluripotency factors SOX2 and NANOG in BGC-823 cells cultured with different conditioned media was also examined. RESULTS: GCAFs displayed higher capacities to induce BGC-823 cell proliferation, migration, and invasion than GNFs (P < 0.01). Astragaloside IV treatment strongly inhibited the proliferation-, migration- and invasion-promoting capacities of GCAFs (P < 0.05 for 10 µmol/L, P < 0.01 for 20 µmol/L and 40 µmol/L). Compared with GNFs, GCAFs expressed a lower level of microRNA-214 (P < 0.01) and a higher level of microRNA-301a (P < 0.01). Astragaloside IV treatment significantly up-regulated microRNA-214 expression (P < 0.01) and down-regulated microRNA-301a expression (P < 0.01) in GCAFs. Reestablishing the microRNA expression balance subsequently suppressed M-CSF production (P < 0.01) and secretion (P < 0.05), and elevated TIMP2 production (P < 0.01) and secretion (P < 0.05). Consequently, the ability of GCAFs to increase SOX2 and NANOG expression in BGC-823 cells was abolished by astragaloside IV. CONCLUSION: Astragaloside IV can inhibit the pathological functions of GCAFs by correcting their dysregulation of microRNA expression, and it is promisingly a potent therapeutic agent regulating tumor microenvironment.

[Effect of Tongxinluo on Apoptosis of Rat Cardiac Microvascular Endothelial Cells].

OBJECTIVE: To observe the protective effects of Tongxinluo (TXL) on apoptosis of rat cardiac microvascular endothelial cells (RCMECs) resulting from homocysteine (Hcy) induced endoplasmic reticulum stress (ERS), and to determine the signaling pathway behind its protection. METHODS: Primary cultured RCMECs were isolated from neonatal rats using tissue explant method. The morphology of RCMECs was observed using inverted microscope, identified and differentiated by CD31 immunofluorescence method. Selected were well growing 2nd-4th generations of RCMECs. The optimal action time was determined by detecting the expression of glucose regulated protein 78 (GRP78) using immunofluorescence method. In the next experiment RCMECs were divided into 5 groups, i.e., the blank control group, the Hcy induced group (Hcy 10 mmol/L, 10 h), the Hcy + TXL group (Hcy 10 mmol/L + TXL 400 µg/mL), the Hcy +LY294002 group (Hcy 10 mmol/L + LY294002 5 µmol/L, LY294002 as the inhibitor of PI3K), the Hcy + LY294002 + TXL group (Hcy 10 mmol/L + LY294002 5 µmol/L + TXL 400 µg/mL). The apoptosis rate of RCMECs was detected by flow cytometry. mRNA and protein expressions of GRP78, C/ EBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase12) were detected by real-time reverse transcription PCR (RT-PCR) and Western blot respectively. Expression levels of phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T- P13K) , phosphorylation of kinase B (P-Akt) , and total kinase B (T-Akt) were detected by Western blot. RESULTS: Ten hours Hcy action time was determined. Compared with the blank control group, the apoptosis rate was increased (22.77%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, protein expressions of P-PI3K and P-Akt,ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy induced group (P < 0.05, P < 0.01). Compared with the Hcy induced group, the apoptosis rate was decreased (10.17%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were decreased, and expression levels of P-PI3K, P-Akt, P-PI3K/T-PI3K, and P-Akt/T-Akt were increased in the Hcy + TXL group (P < 0.05, P < 0.01). Compared with the Hcy + TXL group, the apoptosis rate was increased (17.9%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, expression levels of P-PI3K and P-Akt, ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy + TXL + LY294002 group (P < 0.05, P < 0.01). CONCLUSION: TXL could inhibit the apoptosis of RCMECs resulting from Hcy-induced ERS and its mechanism might be associated with activating PI3K/Akt signaling pathway.

[Synthesis and characterization of 9-O-alkyl substituted palmatine derivatives].

For the establishment of chemical library of protoberberines provided for the bio-activity screening, the target compounds were synthesized by thermal degradation and nucleophilic substitution reactions with the bio-active alkaloid, palmatine (1), as the raw material, and their structures were identified and conformed by 1H-NMR and MS spectra. Among them, 13 compounds were new.

[The effect of baicalin on human brain microvascular endothelial cell under the glucose deprivation combined with hypoxia condition].

OBJECTIVE: To investigate the mechanisms of baicalin on anti-cerebral ischemic through observing the effect of baicalin on human brain microvascular endothelial cell under the glucose deprivation combined with hypoxia condition. METHODS: Human brain microvascular endothelial cells (HBMVECs) cultured in vitro were divided into the following groups: normal group, model group, baicalin high dose group, baicalin middle dose group, baicalin low dose group, nimodipine group. The kits were used to detect the cell viability, leakage rate of lactate dehydrogenase (LDH), Na(+)-K(+)-ATPase, Ca(2+)-ATPase, the concentration of Ca2+ in each group, and apoptosis rates of each group were analyzed by flow cytometry (FCM). RESULTS: Compared with the normal group, the cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase in model group decreased significantly (P < 0.01, P < 0.05). But the leakage rate of LDH, Ca2+ in cells and apoptosis rates increased remarkably (P < 0.01). Compared with the model group, the cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase increased obviously (P < 0.01, P < 0.05) in baicalin high dose group. But the leakage rate of LDH and Ca2+ in cells in baicalin high dose group decreased significantly comparing with that of model group (P < 0.05, P < 0.01). And the reduction of intracellular Ca2+ was superior to that of nimodipine group. Meanwhile, the apoptosis rates decreased significantly in both baicalin high and middle dose groups (P < 0.01). CONCLUSION: Baicalin could improve the cell viability of HBMVECs under the glucose deprivation combined with hypoxia condition. And the mechanisms were related with improving the energy metabolism, inhibiting intracellular calcium overload and decreasing the apoptosis rate of cells further.

Chinese medicine Tongxinluo modulates vascular endothelial function by inducing eNOS expression via the PI-3K/Akt/HIF-dependent signaling pathway.

AIM OF THE STUDY: To investigate the molecular mechanisms whereby the Chinese medicinal compound Tongxinluo improves vascular endothelial function through studying the induction of endothelial nitric oxide synthase (eNOS) and its upstream signaling pathway. MATERIALS AND METHODS: Hyperhomocysteinemia was induced in Wistar rats by a methionine-rich diet followed by Tongxinluo treatment. The aorta ring was isolated for measuring vascular dilation of aorta and eNOS expression. Human umbilical vein endothelial cells (HUVECs) were transfected with AP-1, NF-κB, HRE or eNOS reporter plasmid followed by Tongxinluo exposure. Expression of the reporter genes was measured by luciferase assay. The level of eNOS was studied by western blot and the nitric oxide content was measured using the nitrate reductase method. HUVECs were also transiently transfected with the dominant negative mutant of HIF-1, PI-3K or Akt to explore the role of HIF and PI-3K/Akt pathway in eNOS induction by Tongxinluo. RESULTS: Tongxinluo could significantly up-regulate the expression of eNOS in the aortic tissue and improve the endothelium-dependent vasodilation of the aorta ring. Additionally, Tongxinluo at various doses could significantly enhance the expression of HRE and eNOS reporter gene as well as up-regulate the protein level of eNOS. Meanwhile, Tongxinluo caused a dose-dependent increase in the NO content in the supernatant of HUVECs. Suppression of HIF-1 activation by DN-HIF or inhibition of PI-3K/Akt pathway by ΔP85 or DN-Akt both attenuated HRE reporter gene activation and eNOS induction by Tongxinluo. CONCLUSION: Tongxinluo, a compound Chinese traditional medicine, up-regulates the expression of eNOS via the PI-3K/Akt/HIF-dependent signaling pathway, thus improving the endothelium-dependent vasodilation.

Effect of serum from overfatigue rats on JNK/c-Jun/HO-1 pathway in human umbilical vein endothelial cells and the intervening effect of Tongxinluo superfine powder.

OBJECTIVE: To cultivate human umbilical vein endothelial cells (HUVECs) in the serum of overfatigue rats with the intervention of Tongxinluo superfine powder (TXLSP). By examining the variation of the activity of JNK/c-Jun/HO-1 pathway, the possible mechanisms of vascular endothelial dysfunction under overfatigue conditions and the intervening effect of TXLSP were explored. METHODS: The HUVECs were randomly divided into the normal control group, the model group, the SP600125 (a specific antagonist of JNK) group, the TXLSP group and the TXLSP + SP600125 group. The content of carboyhemoglobin (COHb) and the leak rate of lactic dehydrogenase (LDH) in different groups were measured. The mRNA and protein expression of JNK, c-Jun, HO-1 and the phosphorylation level of c-Jun (P-c-Jun) were detected using Western blot and PCR methods. RESULTS: Compared with the normal control group, the COHb level in supernatant was increased significantly in the model group, and the expression of HO-1, JNK, c-Jun mRNA and corresponding proteins and P-c-Jun were also increased remarkably. The increases in these parameters were significantly decreased by SP600125. TXLSP showed remarkable up-regulation on the expression of JNK, c-Jun, P-c-Jun and HO-1 mRNA and their protein expression. Compared with the SP600125 group, the expressions of JNK, c-Jun, P-c-Jun and HO-1 mRNA and its protein in the TXLSP+SP600125 group were significantly increased at different time points (P<0.05, P<0.01). CONCLUSIONS: The vascular endothelial dysfunction under overfatigue conditions is related to the activity of the JNK/c-Jun/HO-1 pathway. One of the mechanisms of TXLSP in improving the vascular endothelial function is to adjust the activity of the JNK/c-Jun/HO-1 pathway at gene and protein levels.

[The endothelium injuries caused by homocysteine and treatmental effects of Tongxinluo powder].

AIM: To observe the effect of homocysteine (HCY) on the function of endothelium cell, and to discuss the possible mechanisms that Tongxinluo super powder affected. METHODS: Healthy male Wistar rats were divided into randomly the control group, the model group, the Tongxinluo group. The effect of Ach on isolated rat thoracic aorta in vitro was examined, the microcirculation was observed by microcirculation meter, the activity of SOD and GSH-PX and content of NO, MDA, ET, Ang II, TXA2, PGI2 was detected. RESULTS: Compared with control group, the effect of Ach on isolated rat thoracic aorta in vitro weakened markablely (P < 0.01), the format and percentage that capillary dilated declined significantly (P < 0.05), after treatment with Tongxinluo powder, the effect of Ach on isolated rat thoracic aorta in vitro was improved obviously (p < 0.01), and the format and percentage that capillary dilated were increased compared with model group; comparing with the control group, the level of Ang II and ET, TXA2 in plasm increased obviously (P < 0.05, P < 0.01), while the content of PGI2 depressed manifestly (P < 0.05), at the same time, both content of NO and activity of SOD, (GSH-PX declined obviously (P < 0.001, P < 0.05). After treatment with Tongxinluo powder, the level of ET, AngII and TXA2 reduced significantly in different degree (P < 0.01), while the content of PGI2 appeared stepping up notably (P < 0.01), and both activity of SOD and NO level increased obviously (P < 0.01, P < 0.05). CONCLUSION: (1) The high homocystein might cause the contracted and dilated function decreased, it might get involved in endothelium disfunction as a result of the massive free radicals production and diastolic-contract factors balance disorder induced by high homocystein. (2) Tongxinluo powder could improve the function of endothelium-dependment dilation induced by high homocystein, that associated with inhibitting the excessive production of free radicals, and improved function of endothelium.

[The effects of Tongxinluo Supermicro Powder on nuclear factor-kappaB, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in aorta of rabbits fed with high-lipid diet].

OBJECTIVE: To observe the effects of Tongxinluo Supermicro Powder on the nuclear factor-kappaB (NF-kappaB), inter-cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in aorta of rabbits fed with high-lipid diet. METHODS: Healthy male New Zealand rabbits were randomly divided into 4 groups (n = 8 each): control group, model group, atorvastatin group (3 mg x kg(-1) x d(-1) per gavage), and Tongxinluo group (0.31 g x kg(-1) x d(-1) per gavage). At the end of 6 weeks, the expression of NF-kappaB, ICAM-1 and VCAM-1 were observed by immunochemistry methods, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). RESULT: The nuclear translocation of NF-kappaB in aortic endothelial cells and the gene expressions of NF-kappaB, ICAM-1 and VCAM-1 at protein and mRNA levels of the model group was significantly increased compared that in the control group (all P < 0.05), these effects could be significantly attenuated by atorvastatin and Tongxinluo Supermicro Powder (P < 0.01 vs. model group). CONCLUSIONS: Similar as atorvastatin, Tongxinluo Supermicro Powder could relieve the process of atherosclerosis by decreasing the nuclear translocation of NF-kappaB and reducing the expression of ICAM-1, VCAM-1 in this model.

Enriched selenium and its effects on growth and biochemical composition in Lactobacillus bulgaricus.

Se-enriched Lactobacillus bulgaricus (L. bulgaricus) was generated by administration of sodium selenite (0, 1, 4, 8, 16, 32, and 64 mg/L, respectively) in MRS medium and enriched selenium manifestation in L. bulgaricus was investigated using transmission electron microscopy and energy-dispersive X-ray spectrometry and alterations of essential elements and amino acids in the organism were evaluated. We demonstrate that administration of sodium selenite in the dosage of 1-16 mg/L is suitable for selenium enrichment in L. bulgaricus and can enhance nutritive value in the organism by elevating the contents of essential elements including P, Mg, Mn, Zn, Ca, and total amino acids as well as reducing selenite to insoluble elemental selenium, an electron-dense and amorphous Se (0) granule, thereby depositing it both in the cytoplasm and in the extracellular space of L. bulgaricus. Thus, Se-enriched Lactobacillus can provide a potential dietary source of nontoxic selenium and functional regulator used for food and medical industry.