D-beta-hydroxybutyrate protects against microglial activation in lipopolysaccharide-treated mice and BV-2 cells.

Microglial activation is a key event in neuroinflammation, which, in turn, is a central process in neurological disorders. In this study, we investigated the protective effects of D-beta-hydroxybutyrate (BHB) against microglial activation in lipopolysaccharide (LPS)-treated mice and BV-2 cells. The effects of BHB in mice were assessed using behavioral testing, morphological analysis and immunofluorescence labeling for the microglial marker ionizing calcium-binding adaptor molecule 1 (IBA-1) and the inflammatory cytokine interleukin-6 (IL-6) in the hippocampus. Moreover, we examined the levels of the inflammatory IL-6 and tumor necrosis factor-α (TNF-α), as well as those of the neuroprotective brain-derived neurotrophic factor (BDNF) and transforming growth factor-ß (TGF-ß) in the brain. In addition, we examined the effects of BHB on IL-6, TNF-α, BDNF, TGF-ß, reactive oxygen species (ROS) level and cell viability in LPS-stimulated BV-2 cells. BHB treatments attenuated behavioral abnormalities, reduced the number of IBA-1-positive cells and the intensity of IL-6 fluorescence in the hippocampus, with amelioration of microglia morphological changes in the LPS-treated mice. Furthermore, BHB inhibited IL-6 and TNF-α generation, but promoted BDNF and TGF-ß production in the brain of LPS-treated mice. In vitro, BHB inhibited IL-6 and TNF-α generation, increased BDNF and TGF-ß production, reduced ROS level, ameliorated morphological changes and elevated cell viability of LPS-stimulated BV-2 cells. Together, our findings suggest that BHB exerts protective effects against microglial activation in vitro and in vivo, thereby reducing neuroinflammation.

Taurine inhibits KDM3a production and microglia activation in lipopolysaccharide-treated mice and BV-2 cells.

Microglia activation has been suggested as the key factor in neuro-inflammation and thus participates in neurological diseases. Although taurine exhibits anti-inflammatory and neuro-protective effects, its underlying epigenetic mechanism is unknown. In this study, taurine was administered to lipopolysaccharide (LPS)-treated mice and BV-2 cells. Behavioral test, morphological analyze, detection of microglia activation, and lysine demethylase 3a (KDM3a) measurements were performed to investigate the mechanism by which taurine regulates KDM3a and subsequently antagonizes microglia activation. Taurine improved the sociability of LPS-treated mice, inhibited microglia activation in the hippocampus, and reduced generation of brain inflammatory factors, such as interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase, and cyclooxygenase-2. Meanwhile, taurine suppressed the LPS-induced increase in microglial KDM3a, and increased the level of mono-, di- or tri-methylation of lysine 9 on histone H3 (H3K9me1/2/3). Furthermore, taurine inhibited the LPS-induced increase in KDM3a, elevated the H3K9me1/2/3 level, and reduced inflammatory factors and reactive oxygen species in a concentration-dependent manner in LPS-stimulated BV-2 cells. In conclusion, taurine inhibited KDM3a and microglia activation, thereby playing an anti-inflammatory role in LPS-treated mice and BV-2 cells.

ß-Hydroxybutyrate inhibits histone deacetylase 3 to promote claudin-5 generation and attenuate cardiac microvascular hyperpermeability in diabetes.

AIMS/HYPOTHESIS: Microvascular endothelial hyperpermeability, mainly caused by claudin-5 deficiency, is the initial pathological change that occurs in diabetes-associated cardiovascular disease. The ketone body ß-hydroxybutyrate (BHB) exerts unique beneficial effects on the cardiovascular system, but the involvement of BHB in promoting the generation of claudin-5 to attenuate cardiac microvascular hyperpermeability in diabetes is poorly understood. METHODS: The effects of BHB on cardiac microvascular endothelial hyperpermeability and claudin-5 generation were evaluated in rats with streptozotocin-induced diabetes and in high glucose (HG)-stimulated human cardiac microvascular endothelial cells (HCMECs). To explore the underlying mechanisms, we also measured ß-catenin nuclear translocation, binding of ß-catenin, histone deacetylase (HDAC)1, HDAC3 and p300 to the Claudin-5 (also known as CLDN5) promoter, interaction between HDAC3 and ß-catenin, and histone acetylation in the Claudin-5 promoter. RESULTS: We found that 10 weeks of BHB treatment promoted claudin-5 generation and antagonised cardiac microvascular endothelial hyperpermeability in rat models of diabetes. Meanwhile, BHB promoted claudin-5 generation and inhibited paracellular permeability in HG-stimulated HCMECs. Specifically, BHB (2 mmol/l) inhibited HG-induced HDAC3 from binding to the Claudin-5 promoter, although nuclear translocation or promoter binding of ß-catenin did not change with BHB treatment. In addition, BHB prevented the binding and co-localisation of HDAC3 to ß-catenin in HG-stimulated HCMECs. Furthermore, using mass spectrometry, acetylated H3K14 (H3K14ac) in the Claudin-5 promoter following BHB treatment was identified, regardless of whether cells were stimulated by HG or not. Although reduced levels of acetylated H3K9 in the Claudin-5 promoter were found following HG stimulation, increased H3K14ac was specifically associated with BHB treatment. CONCLUSIONS/INTERPRETATION: BHB inhibited HDAC3 and caused acetylation of H3K14 in the Claudin-5 promoter, thereby promoting claudin-5 generation and antagonising diabetes-associated cardiac microvascular hyperpermeability. Graphical abstract.

C-peptide prevents NF-κB from recruiting p300 and binding to the inos promoter in diabetic nephropathy.

C-peptide (CP) has demonstrated unique beneficial effects in diabetic nephropathy (DN), but whether and how CP regulates NF-κB and its coactivator, p300, to suppress inducible iNOS and antagonize DN are unknown. iNOS expression, NF-κB nuclear translocation, colocalization and binding of NF-κB to p300, binding of NF-κB to the inos promoter, and the bound NF-κB, p300, and histone 3 lysine 9 acetylation (H3K9ac) at binding sites were measured in high glucose-stimulated mesangial cells. We evaluated pathologic changes, iNOS expression, NF-κB, and p300 contents in diabetic rats. We found that CP inhibited iNOS expression and notably prevented colocalization and binding of NF-κB and p300. CP prevented NF-κB from binding to the inos promoter, especially at the distal site, and reduced bound NF-κB, p300, and H3K9ac. N-terminal plus middle fragment could mostly mimic the antagonizing effects of CP against the pathologic changes of DN and equally suppresses renal iNOS expression as CP. In conclusion, CP prevented NF-κB from recruiting p300 and binding to the inos promoter, and decreased H3K9ac at the binding sites to suppress iNOS expression and antagonize DN, with the effect region identified as N-terminal plus middle fragment.-Li, Y., Li, X., He, K., Li, B., Liu, K., Qi, J., Wang, H., Wang, Y., Luo, W. C-peptide prevents NF-κB from recruiting p300 and binding to the inos promoter in diabetic nephropathy.

High glucose decreases claudins-5 and -11 in cardiac microvascular endothelial cells: Antagonistic effects of tongxinluo.

Purpose/aim of the study: Claudins-5, -9, and -11 are tight-junction proteins that are mainly expressed in endothelial cells. Their deficiency may lead to cell barrier dysfunction, which is considered as the initiating process and pathological basis of cardiovascular disease in diabetes. We investigated whether high glucose (HG) affects claudins-5, -9, and -11 in human cardiac microvascular endothelial cells (HCMECs), and examined the effects of the traditional Chinese medication tongxinluo (TXL) on these tight junction proteins. MATERIALS AND METHODS: HCMECs were exposed to HG with and without TXL treatment, and then mRNA and protein levels of claudins-5, -9, and -11 were examined. The distribution of claudins-5 and -11 was also investigated. Histone H3K9 acetylation (H3K9ac) in claudin-5 and claudin-11 gene promoters, which functions in transactivation, was measured. RESULTS: We found that HG suppressed claudins-5 and -11 gene expression in HCMECs, and TXL reversed the HG-mediated inhibition of claudins-5 and -11 mRNA and protein expressions. Treatment with high-dose of TXL promoted cell membrane localization of claudins-5 and -11 in HG-stimulated HCMECs. Furthermore, high-dose of TXL blocked the inhibition of H3K9ac in claudin-5 and claudin-11 gene promoters caused by exposure to HG, thus activating gene transcription. CONCLUSIONS: Our results show that HG suppressed claudins-5 and -11 in HCMECs, and TXL could reverse the HG-induced suppression of claudins-5 and -11 by increasing H3K9ac in their respective gene promoters.

Developmental Stage-Specific Embryonic Induction of HepG2 Cell Differentiation.

BACKGROUND: Although hepatocellular carcinoma cells can sometimes undergo differentiation in an embryonic microenvironment, the mechanism is poorly understood. AIM: The developmental stage-specific embryonic induction of tumor cell differentiation was investigated. METHODS: Both chick and mouse liver extracts and hepatoblast-enriched cells at different developmental stages were used to treat human hepatoma HepG2 cells, and the effects on the induction of differentiation were evaluated. The nuclear factors controlling differentiation, hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and upstream stimulatory factor-1 (USF-1), and the oncogene Myc and alpha-fetoprotein (AFP) were measured. HNF-4α RNA interference was used to verify the role of HNF-4α. Embryonic induction effects were further tested in vivo by injecting HepG2 tumor cells into immunodeficient nude mice. RESULTS: The 9-11-days chick liver extracts and 13.5-14.5-days mouse hepatoblast-enriched cells could inhibit proliferation and induce differentiation of HepG2 cells, leading to either death or maturation to hepatocytes. The maturation of surviving HepG2 cells was confirmed by increases in the expressions of HNF-4α, HNF-1α, HNF-6, and USF-1, and decreases in Myc and AFP. The embryonic induction of HepG2 cell maturation could be attenuated by HNF-4α RNA interference. Furthermore, the 13.5-days mouse hepatoblast culture completely eliminated HepG2 tumors with inhibited Myc and induced HNF-4α, confirming this embryonic induction effect in vivo. CONCLUSIONS: This study demonstrated that developmental stage-specific embryonic induction of HepG2 cell differentiation might help in understanding embryonic differentiation and oncogenesis.

Developmental Stage-Specific Hepatocytes Induce Maturation of HepG2 Cells by Rebuilding the Regulatory Circuit.

On the basis of their characteristics, we presume that developmental stage-specific hepatocytes should have the ability to induce maturation of hepatoma cells. A regulatory circuit formed by hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and the upstream stimulatory factor (USF-1) play a key role in the maturation of embryonic hepatocytes; however, it is unclear whether the regulatory circuit mediates the embryonic induction of hepatoma cell maturation. In this study, 12.5-d to 15.5-d mouse embryonic hepatocytes or their medium were used to coculture or treat HepG2 cells, and the induced maturation was evaluated in vitro and in vivo. In the induced HepG2 cells, the components of the regulatory circuit were detected, their cross-regulation was evaluated and HNF-4α RNA interference was performed. We found that 13.5-d to 14.5-d embryonic hepatocytes could induce HepG2 cell maturation, demonstrated by morphological changes, increased maturation markers and decreased c-Myc and α-fetoprotein (AFP) in vitro. The majority of HepG2 tumors were eliminated by 13.5-d embryonic induction in vivo. All components of the regulatory circuit were upregulated and the binding of HNF-4α, HNF-1α, HNF-6 and USF-1 to their target sites was promoted to rebuild the regulatory circuit in the induced HepG2 cells. Moreover, RNA interference targeting HNF-4α, which is the core of the regulatory circuit, attenuated the induced maturation of HepG2 cells with downregulation of the regulatory circuit. These results revealed that developmental stage-specific hepatocytes could induce the maturation of HepG2 cells by rebuilding the regulatory circuit.

D-beta-hydroxybutyrate reduced the enhanced cardiac microvascular endothelial FoxO1 to play protective roles in diabetic rats and high glucose-stimulated human cardiac microvascular endothelial cells.

The O subfamily of forkhead (FoxO) 1 may participate in the pathogenesis of diabetic microvascular endothelial injury. However, it is unknown whether D-beta-hydroxybutyrate (BHB) regulates cardiac microvascular endothelial FoxO1 to play protective roles in diabetes. In the study, limb microvascular morphological changes, endothelial distribution of the tight junction protein Claudin-5 and FoxO1, and FoxO1 content in limb tissue from clinical patients were evaluated. Then the effects of BHB on cardiac microvascular morphological changes, cardiac FoxO1 generation and its microvascular distribution in diabetic rats were measured. And the effects of BHB on FoxO1 generation in high glucose (HG)-stimulated human cardiac microvascular endothelial cells (HCMECs) were further analyzed. The results firstly confirmed the enhanced limb microvascular FoxO1 distribution, with reduced Claudin-5 and endothelial injury in clinical patients. Then the elevated FoxO1 generation and its enhanced cardiac microvascular distribution were verified in diabetic rats and HG-stimulated HCMECs. However, BHB inhibited the enhanced cardiac FoxO1 generation and its microvascular distribution with attenuation of endothelial injury in diabetic rats. Furthermore, BHB reduced the HG-stimulated mRNA expression and protein content of FoxO1 in HCMECs. In conclusion, BHB reduced the enhanced cardiac microvascular endothelial FoxO1 to play protective roles in diabetic rats and HG-stimulated HCMECs.

Crocin ameliorates atherosclerosis by promoting the reverse cholesterol transport and inhibiting the foam cell formation via regulating PPARγ/LXR-α.

Crocin (CRO) is feasible in alleviating atherosclerosis (AS), the mechanism of which was therefore explored in the study. High-fat diet (HFD)-induced apolipoprotein E-deficient (ApoE-/-) mice and lysophosphatidic acid (LPA)-treated macrophages received CRO treatment. Treated macrophage viability was determined via MTT assay. In both murine and macrophage, the lipid level and total Cholesterol/Cholesteryl l Ester (TC/CE) levels were quantified by oil-red-O staining and ELISA, respectively. Lipid droplet, aortic plaque formation and collagen deposition were detected via Oil-red-O staining, hematoxylin-eosin staining and Masson staining, respectively. Liver X Receptor-α (LXR-α), Peroxisome Proliferator-Activated Receptor γ (PPARγ), CD68, PCSK9, CD36, ATP Binding Cassette Subfamily A Member 1 (ABCA1), phosphorylated (p)-AKT, and AKT expressions were detected via Western blot, the former three also being detected using Immunohistochemistry and the first being measured by qRT-PCR. CRO decreased HFD-induced weight gain, ameliorated the abnormal serum lipid levels of HFD-treated mice, and inhibited aortic plaque formation and lipid deposition, and increased collagen fibers, with upregulated high-density lipoprotein-cholesterol (HDL-C) and downregulated TC and low-density lipoprotein-cholesterol (LDL-C). CRO alleviated the HFD-induced upregulations of CD68, PCSK9 and CD36 as well as downregulations of PPARγ/LXR-α, ABCA1 and AKT phosphorylation. In LPA-treated macrophages, CRO alone exerted no effect on the viability yet inhibited the lipid droplets formation and downregulated TC/CE levels. Silent LXR-α reversed the effect of CRO on the lipid droplets formation and levels of lipid metabolism-related factors. CRO ameliorated AS by inhibiting foam cells formation and promoting reverse cholesterol transport via PPARγ/LXR-α.

Prebiotics and oxidative stress in constipated rats.

OBJECTIVES: Constipation can adversely affect children's health, with disorders of host immunity and enhanced oxidative stress. As nondigestible carbohydrates, prebiotics can affect the host with constipation; however, whether the prebiotics have effects on the content of intestinal secretory immunoglobulin A (sIgA) and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in constipation has not been fully clarified. METHODS: In the present study, constipation was induced in female Sprague-Dawley rats by diphenoxylate, and the prebiotics dissolved in milk were used as an intervention. The indicators of intestinal peristalsis, including the time of passing black stool initially, the grains of black stool in 24 hours, and the advance rate of ponceau, were measured. The content of intestinal sIgA was detected by enzyme-linked immunosorbent assay. The contents of SOD and MDA in serum and intestinal tissue were analyzed by their detection kits. RESULTS: The changes in intestinal peristalsis show obvious constipation. The content of intestinal sIgA decreases, the content of SOD decreases, but the content of MDA increases in constipated rats. Prebiotics can attenuate the constipation-caused abnormal indicators significantly. CONCLUSIONS: Prebiotics can attenuate decreased intestinal immunity and enhanced oxidative stress, in addition to reduced intestinal peristalsis and of the constipated rats.

ß-Hydroxybutyrate inhibits cardiac microvascular collagen 4 accumulation by attenuating oxidative stress in streptozotocin-induced diabetic rats and high glucose treated cells.

Accumulation of collagen 4 (COL4) and thickened basement membrane are features of diabetic cardiac microvascular fibrosis that may be induced by oxidative stress. The ketone body ß-hydroxybutyrate exhibits various cardiovascular protective effects, however its mechanism remains to be clarified. In the current study, the effects of ß-hydroxybutyrate on cardiac microvascular fibrosis and COL4 accumulation were evaluated in streptozotocin-induced diabetic rats and in high glucose (HG) treated human cardiac microvascular endothelial cells (HCMECs). Generations of inducible nitric oxide synthase (iNOS) and copper-zinc superoxide dismutase (Cu/Zn-SOD), and the amount of nitrotyrosine (NT) were measured in vivo and in vitro. Ten weeks of ß-hydroxybutyrate treatment (160, 200 and 240 mg/kg/d) attenuated cardiac microvascular fibrosis and inhibited cardiac COL4 generation and microvascular distribution in diabetic rats. Furthermore, ß-hydroxybutyrate promoted cardiac Cu/Zn-SOD generation and reduced NT content, without reducing iNOS generation in diabetic rats. In HCMECs, stimulation with HG induced excess generation of COL4 via peroxynitrite. ß-Hydroxybutyrate treatment (2, 4, 6 mM) attenuated HG-stimulated COL4 accumulation in a concentration-dependent manner. Similarly, 4 mM ß-hydroxybutyrate promoted Cu/Zn-SOD generation and reduced NT content, without affecting excess iNOS generation in HG-stimulated HCMECs. In conclusion, this study showed that ß-hydroxybutyrate promoted Cu/Zn-SOD generation, reduced peroxynitrite and inhibited cardiac microvascular COL4 accumulation in diabetes.

Peroxynitrite-induced nitration of cyclooxygenase-2 and inducible nitric oxide synthase promotes their binding in diabetic angiopathy.

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) play crucial roles in diabetic angiopathy. In vivo, however, the following facts remain unknown: whether COX-2 and iNOS bind, how peroxynitrite-induced nitration of COX-2 and iNOS affects their binding if they do bind and what effects of this mechanism contribute to diabetic angiopathy. This study focused on the issues above. Diabetes was induced in Wistar male rats by intraperitoneal injection of streptozotocin. As a specific scavenger of peroxynitrite, urate was used. After 13 wks of diabetes, the morphological and biochemical changes of the rats showed obvious diabetic angiopathy. There exists in vivo colocalization and binding of COX-2 and iNOS in diabetic angiopathy. The nitration level of total and co-immunoprecipitated COX-2 and iNOS increased significantly, and, simultaneously, their binding and activity increased in the diabetes group. In the diabetes + urate group, the nitration level of COX-2 and iNOS decreased and their binding reduced, consistent with their decreased activity and the attenuated pathological changes in the rat aorta and glomerulus. The results provide in vivo evidence that COX-2 and iNOS can bind in diabetic angiopathy and that peroxynitrite-induced nitration of COX-2 and iNOS promotes their binding, contributing to diabetic angiopathy.

Tumor supernatant derived from hepatocellular carcinoma cells treated with vincristine sulfate have therapeutic activity.

Vincristine sulfate (VCR), a commonly used chemotherapeutic agent, kills cancer cells as well as the normal cells for its cytotoxicity. But it is still unclear whether it can exert therapeutic effect on untreated cancer cells by changing the supernatant of cancer cells. Here, we explored the subsequent cascade effects of the supernatant of cancer cells that were transiently treated with VCR on untreated tumor cells and its responsible mechanisms. VCR and three different hepatocellular carcinoma (HCC) cell lines were used for an experiment. The experiment was conducted in vitro to eliminate the body's internal factors and the effects of the immune system. The results suggested that drug-free tumor supernatant (TSN) could promote the differentiation, repress the transcription of liver cancer stem cell's markers and the proliferation in SMMC-7721, Bel-7402 and Huh7 cells. Furthermore, we found that the TSN could abolish YAP1 transcriptional activity to inhibit the proliferation and increase the transcriptional activity of HNF4α to promote the differentiation in SMMC-7721 and Bel-7402 cells. In conclusion, the TSN could inhibit the proliferation and induce differentiation in different HCC cells.

Up-regulation of MMP-2 by histone H3K9 ß-hydroxybutyrylation to antagonize glomerulosclerosis in diabetic rat.

AIMS: Besides energy supply, ß-hydroxybutyrate (BHB) acts as a bioactive molecule to play multiple protective roles, even in diabetes and its complications. The aim of this study was to investigate the antagonizing effects of BHB against diabetic glomerulosclerosis and the underlying mechanism. METHODS: Male Sprague-Dawley rats were intraperitoneally injected with streptozotocin to induce diabetes and then treated with different concentrations of ß-hydroxybutyrate. After 10 weeks, body weight, blood glucose, serum creatinine and 24-h urine protein were examined. Glomerular morphological changes and the contents of collagen type IV (COL IV) were evaluated. Then, transforming growth factor (TGF)-ß/Smad3 contents and matrix metalloproteinase-2 (MMP-2) generation were detected. Moreover, the total contents of trans-activating histone H3K9 ß-hydroxybutyrylation (H3K9bhb) and the contents of H3K9bhb in the Mmp-2 promoter were measured. RESULTS: It was firstly confirmed that BHB treatments reduced renal biochemical indicators and attenuated glomerular morphological changes of the diabetic rats, with COL IV content decreased in a concentration-dependent manner. Then, BHB treatments were found to up-regulate renal MMP-2 generation of the diabetic rats significantly, while not affecting the increased TGF-ß/Smad3 contents. Furthermore, the contents of H3K9bhb in the Mmp-2 promoter were elevated significantly for the middle and high concentrations of BHB treatments, up-regulating MMP-2 generation. CONCLUSION: BHB treatments could up-regulate MMP-2 generation via causing elevated H3K9bhb in its promoter to antagonize glomerulosclerosis in the diabetic rats.

ß-hydroxybutyrate antagonizes aortic endothelial injury by promoting generation of VEGF in diabetic rats.

Endothelial injury is regarded as the initial pathological process in diabetic vascular diseases, but effective therapy has not yet been identified. Although ß-hydroxybutyrate plays various protective roles in the cardiovascular system, its ability to antagonize diabetic endothelial injury is unclear. ß-hydroxybutyrate reportedly causes histone H3K9 ß-hydroxybutyrylation (H3K9bhb), which activates gene expression; however, there has been no report regarding the role of H3K9bhb in up-regulation of vascular endothelial growth factor (VEGF), a crucial factor in endothelial integrity and function. Here, male Sprague-Dawley rats were intraperitoneally injected with streptozotocin to induce diabetes, and then treated with different concentrations of ß-hydroxybutyrate. After 10 weeks, body weight, blood glucose, morphological changes and serum nitric oxide concentration were examined. Moreover, the mRNA expression level, protein content and distribution of VEGF in the aorta were investigated, as were total protein ß-hydroxybutyrylation and H3K9bhb contents. The results showed injury of aortic endothelium, along with reductions of the concentration of nitric oxide and generation of VEGF in diabetic rats. However, ß-hydroxybutyrate treatment attenuated diabetic injury of the endothelium and up-regulated the generation of VEGF. Furthermore, ß-hydroxybutyrate treatment caused marked total protein ß-hydroxybutyrylation and significant elevation of H3K9bhb content in the aorta of diabetic rats. The ability of ß-hydroxybutyrate to protect against diabetic injury of the aortic endothelium was greatest for its intermediate concentration. In conclusion, moderately elevated ß-hydroxybutyrate could antagonize aortic endothelial injury, potentially by causing H3K9bhb to promote generation of VEGF in diabetic rats.

Peroxynitrite plays a key role in glomerular lesions in diabetic rats.

BACKGROUND: Oxidative stress plays a crucial role in chronic complications of diabetes such as diabetic nephropathy (DN), the main cause of renal failure. In diabetes, whether peroxynitrite (ONOO(-)), generated from inducible nitric oxide synthase (iNOS) induced by tumor necrosis factor alpha (TNF-alpha), plays a primary role in the pathogenesis of glomerular lesion is not yet fully known. This study was designed to investigate the role of exaggerated ONOO(-) in glomerular lesions of diabetic rats. METHODS: Diabetes was induced in Sprague Dawley rats by an intraperitoneal injection of streptozocin, and aminoguanidine was used as selective inhibitor of iNOS. The iNOS transcription and protein distribution and content in rat glomeruli were detected. Nitrotyrosine (NT), a specific marker of ONOO(-), was measured to represent the distribution and content of ONOO(-) in rat glomeruli. TNF-alpha level and nitric oxide (NO) content were evaluated, and the pathological changes in the rat glomeruli were observed. Biochemical indicators of renal function were also measured. RESULTS: TNF-alpha level and NO content, iNOS expression and its protein content, and NT content increased significantly, in accordance with the pathological changes of glomerulus and renal dysfunction in the diabetes group. Aminoguanidine was found to inhibit iNOS and then reduce ONOO(-) overformation, attenuating the pathological alterations. CONCLUSION: This study clarified clearly that exaggerated ONOO(-) formation, generated from induced iNOS may play a key role in glomerular lesions in diabetic rats.

[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.

Targeted induction of bone marrow mesenchymal stem cells to have effectiveness on diabetic pancreatic restoration.

Although bone marrow-derived mesenchymal stem cells (BMSCs) have been reported to be effective for the attenuation of diabetes, they have limitations. Whether BMSCs can be target-induced by pancreatic stem cells (PSCs) to have effectiveness for the restoration of diabetic islet injury was unknown. In this study, based on their successful isolation and cultivation, BMSCs were co-cultured with PSCs. The pancreatic stem cells markers, Nestin and Neurogenin3 in co-cultured BMSCs were detected to evaluate the target-induction effects. After the diabetic rats were intravenously injected with the target-induced BMSCs, general indicators and islet morphology were detected. The islet insulin generation, and serum insulin and C-peptide contents were measured. It was found that after co-culture, the mRNA expressions, protein contents and distributions of Nestin and Neurogenin3, were dramatically high in BMSCs, indicating that they were successfully target-induced to pancreatic stem-like cells. Furthermore, the target-induced BMSCs had beneficial effects on serum glycated albumin levels and glycogen contents as well as islet morphology of the diabetic rats. Besides elevation of islet insulin generation, the target-induced BMSCs had significant effect on serum insulin and C-peptide contents. In conclusion, BMSCs could be target-induced by PSCs to have effectiveness on the pancreatic restoration of diabetic rats.

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.