Tongxinluo Reverses the Hypoxia-suppressed Claudin-9 in Cardiac Microvascular Endothelial Cells.

BACKGROUND: Claudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-11 in hypoxia-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported. METHODS: In this study, HCMECs were stimulated with CoCl2to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin-5, claudin-9, and claudin-11 was confirmed. Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs. RESULTS: We found that hypoxia-suppressed claudin-9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia-suppressed claudin-9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury. Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter (F = 37.766; P = 0.000), activating claudin-9 transcription. CONCLUSIONS: The results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs.

Tongxinluo inhibits cyclooxygenase-2, inducible nitric oxide synthase, hypoxia-inducible factor-2α/vascular endothelial growth factor to antagonize injury in hypoxia-stimulated cardiac microvascular endothelial cells.

BACKGROUND: Endothelial dysfunction is considered as the initiating process and pathological basis of cardiovascular disease. Cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS), inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) are key enzymes with opposing actions in inflammation and oxidative stress, which are believed to be the major driver of endothelial dysfunction. And in hypoxia (Hx), Hx-inducible factor (HIF)-1α and HIF-2α are predominantly induced to activate vascular endothelial growth factor (VEGF), resulting in abnormal proliferation. Whether and how Tongxinluo (TXL) modulates COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF in Hx-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been clarified. METHODS: HCMEC were treated with CoCl 2 to mimic Hx and the mRNA expressions of COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF were first confirmed, and then their mRNA expression and protein content as well as the cell pathological alterations were evaluated for TXL treatment with different concentrations. In addition, the effector molecular of inflammation prostaglandin E 2 (PGE 2 ) and the oxidative marker nitrotyrosine (NT) was adopted to reflect HCMEC injury. RESULTS: Hx could induce time-dependent increase of COX-2, iNOS, HIF-2α, and VEGF in HCMEC. Based on the Hx-induced increase, TXL could mainly decrease COX-2, iNOS, HIF-2α, and VEGF in a concentration-dependent manner, with limited effect on the increase of PGIS and eNOS. Their protein contents verified the mRNA expression changes, which was consistent with the cell morphological alterations. Furthermore, high dose TXL could inhibit the Hx-induced increase of PGE 2 and NT contents, attenuating the inflammatory and oxidative injury. CONCLUSIONS: TXL could inhibit inflammation-related COX-2, oxidative stress-related iNOS, and HIF-2α/VEGF to antagonize Hx-induced HCMEC injury.

[The protective effect of nourishing yin and promoting blood flow recipe on the aortic endothelium injury of diabetic rats].

OBJECTIVE: To investigate the protective effects of Nourishing Yin and Promoting Blood Flow Recipe (NYPBR) on aortic endothelium injury of diabetic rats. METHODS: The SD rats were divided into 3 groups randomly: control group, modle group and NYPBR group. The latter two groups were injected intraperitoneally with streptozotocin to establish diabetes model. Rats in NYPBR group were fed NYPBR solution (3 mL/d), with dose equivalent to the clinical use in the patients. Rats in the other groups were administrated equivalent water. 13 weeks after diabetes was induced, the inducible nitric oxide synthase (iNOS) mRNA expressionin the aorta was detected by RT-PCR, iNOS protein content was determined by Western blotting and immunohistochemistry was used to detect the formation of nitrotyrosine (NT), a specific marker of peroxynitrite (ONOO-). The morphological changes of aortic endothelium were observed under optical microscope and scanning electron microscope (SEM). The levels of endothelin-1 (ET-1) and thromboxane B2 (TXB2) in serum, superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were detected. RESULTS: Compared with control group, the iNOS mRNA expression and its protein content, and the NT content all increased significantly in model group, in accordance with the pathological changes of aortic endothelium. NYPBR improved the pathological alterations obviously. CONCLUSION: NYPBR can decrease iNOS mRNA expression and its protein content, reduce the over-formation of ONOO- and protect the diabetic rats from injury aortic endothelium.

[Protective effect of nourishingyin and promoting blood flow recipe on kidney of diabetic rat].

OBJECTIVE: To investigate the protective effects of Nourishingyin and Promotingblood flow recipe (NYPBR) on the kidney of diabetic rat. METHOD: SD rats were divided into 3 groups at random: control group, diabetes group and NYPBR group. The latter two groups were injected intraperitoneally with streptozotocin to induce diabetes model. Rats in NYPBR group were fed NYPBR solution (3 g x d(-1)), with dose equivalent to the clinical use in the patients. Rats in the other groups were fed equivalent water. 10 weeks after diabetes was induced, the inducible nitric oxide synthase (iNOS) mRNA expression in the renal cortex was detected by RT-PCR, and its protein content by Western blotting. Immunohistochemistry was used to detect the formation of nitrotyrosine (NT), a specific marker of peroxynitrite (ONOO-). The morphological changes of renal cortex were observed under optical microscope. Superoxide dismutase (SOD) and malondialdehyde (MDA) in renal cortex, blood glucose, 24 h urine protein content and creatinine clearance rate in different groups were detected. RESULT: Compared with control group, the iNOS mRNA expression (0.90 +/- 0.10) and its protein content (43.00 +/- 6.08), and NT content (87.23 +/- 5.94) increased significantly in diabetes group, in accord with the pathological changes of renal cortex and renal dysfunction. NYPBR can attenuate the pathological alterations. CONCLUSION: NYPBR could decrease iNOS mRNA expression and its protein content, and reducing the overformation of ONOO-, thus protecting the kidney of diabetic rat from injury.

Interaction between COX-2 and iNOS aggravates vascular lesion and antagonistic effect of ginsenoside.

AIM OF THE STUDY: Ginseng, the root of Panax ginseng C.A.Meyer (Araliaceae), is one of the most widely used Chinese herbs with hypotensive and cardiotonic actions for thousands of years, but the underlying mechanisms have not been well determined. Ginsenoside, the effective components of ginseng, has anti-inflammatory and anti-oxidative effects. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are key enzymes in inflammation and oxidative stress, respectively, which have close interaction, aggravating their damaging effects. This study investigated whether COX-2 interacted with iNOS in vascular endothelial lesion and the protective effect of ginsenoside. MATERIALS AND METHODS: SD male rats were fed with high l-methionine (3%, w/w) to induce vascular endothelial lesion, and the rats in ginsenoside group were fed ginsenoside solution (0.8 mg kg(-1)d(-1)). The mRNA expression and protein contents of COX-2 and iNOS were detected by RT-PCR and Western blotting, respectively. The interaction between COX-2 and iNOS was analyzed by co-immunoprecipitation and laser confocal microscopy. The content of NT, a specific marker of peroxynitrite, was evaluated by Western blotting. The morphological changes of vascular endothelium were observed. RESULTS: Compared with control group, the transcription and protein levels of both COX-2 and iNOS increased obviously and their interaction enhanced significantly in model group, in accord with the increased NT content and the pathological alterations of aorta. In ginsenoside group, all these alterations were attenuated significantly (P < 0.01 or P < 0.05). CONCLUSIONS: It is proved that there exists interaction between COX-2 and iNOS, aggravating endothelial lesion through peroxynitrite and ginsenoside might antagonize their interaction, playing a protective role.

Peroxynitrite-induced protein nitration contributes to liver mitochondrial damage in diabetic rats.

Oxidative stress, especially peroxynitrite (ONOO(-))-mediated oxidative stress, plays a key role in diabetes. Mitochondria, as the generating source of ONOO(-), may also be the major damaging target of ONOO(-), which can cause a series of mitochondrial proteins nitration. Therefore, this study aimed to clarify the relationship between the nitration of entire mitochondrial proteins induced by ONOO(-) and liver mitochondrial structural damage in diabetes. Sprague-Dawley male rats were injected with streptozotocin to induce diabetes. After 10 weeks, transmission electron microscopy was used to observe the ultrastructure of liver mitochondria, and reverse transcription-polymerase chain reaction was used to detect liver inducible nitric oxide synthase (iNOS) mRNA expression. Nitrotyrosine (NT) content and distribution were detected with Western blot analysis and immunohistochemistry. In addition, some biochemical indicators were detected to represent oxidative stress and metabolic disorders. In diabetic rats, increasing levels of iNOS mRNA and NT content (P<.05) were observed, in accord with pathological alterations of the ultrastructure of liver mitochondria. Meanwhile, some alterations in biochemical indicators were observed in diabetes. Treatment with aminoguanidine could significantly attenuate these alterations (P<.01 or P<.05). In conclusion, the nitration of mitochondrial proteins induced by ONOO(-) may be responsible for structural damage to liver mitochondria, and aminoguanidine can reduce ONOO(-) generation and attenuate mitochondrial damage.

[Effect of Bulbus Allii Macrostemi on gene expression profile associated with vascular endothelium injure of Qi stagnation rats].

OBJECTIVE: To investigate the effect of Bulbus Allii Macrostemi on gene expression profile associated with inflammation and oxidative stress in vascular endothelium injure of qi stagnation rats. METHODS: The model of vascular endothelium injure of qi stagnation rats were established by using high L-Methionine and being fastened. RT-PCR and serial analysis of gene expression (SAGE) database, available in NCBI, were used to analyze the changes of genes expression related to inflammation and oxidative stress in endothelium injure and the effect of Bulbus Allii Macrostemi. RESULTS: Compared with model group, the genes expression of inflammation related COX-1 COX-2, oxidative stress related iNOS and eNOS, ECE involving in vascular vasomotion decreased (P < 0.05 or P < 0.01), but the gene expression of antioxidative SOD increased (P < 0.01) in Bulbus Allii Macrostemi group. CONCLUSION: Bulbus Allii Macrostemi can adjust the disorder of the genes expression, related to vascular endothelium injure in Qi stagnation rats, protecting the vascular ehndothelium from injure.

[Effect of Tongxinluo on 7 gene expression profile associated with vascular endothelium injure of rats with deficiency of vital energy or qi stagnation].

OBJECTIVE: To investigate 7 gene expression profile associated with inflammation and oxidative stress in vascular endothelium injure of rats with deficiency of vital energy or qi stagnation, and the effect of Tongxinluo on gene expression profile. METHOD: The model of vascular endothelium injury of rats with deficiency of vital energy or qi stagnation were established by using high L-methionine, with load-carrying swimming or being fastened, respectively. RT-PCR and SAGE database which is available in NCBI, were used to analyze the changes of 7 gene expression related with inflammation and oxidative stress in endothelium injure and the effect of Tongxinluo on the gene expression profile. RESULT: Compared with control group, the gene expression of inflammation related COX-1, COX-2, oxidative stress related iNOS, SOD and blood vessel vasomotion related eNOS, ECE, increased in deficiency of vital energy group (P < 0.05 or P < 0.01), and the gene expression decreased with Tongxinluo treatment (P < 0.05 or P < 0.01). The gene expression of COX-1, COX-2, iNOS and eNOS, ECE, increased (P < 0.01), but the gene expression of PCS and SOD decreased (P < 0.01), in qi stagnation group, and the disorder of gene expression improved with treatment of Tongxinluo (P < 0.01). CONCLUSION: The 7 gene expression related to vascular endothelium injure were not the same in rat with deficiency of vital energy or qi stagnation, and Tongxinluo could regulate the disorder of the gene expression, protecting vascular endothelium from injure.