Lagerstroemia speciosa extract inhibit TNF-induced activation of nuclear factor-κB in rat cardiomyocyte H9c2 cells

Aim of the study

Lagerstroemia speciosa has been used as a folk medicine among people with diabetes in the Philippines. It is known to exhibit antidiabetic, antiobesity, and glucose transport activities through mechanisms not well defined. Diabetes leads to cardiomyocyte hypertrophy in association with an upregulation of vasoactive factors and activation of nuclear factor (NF)-κB and activating protein-1. We therefore investigated the effect of Lagerstroemia speciosa on the activation of NF-κB as a key mediator of cardiomyocyte hypertrophy, in rat cardiomyocyte H9c2 cells.

Materials and methods

Water extract of Lagerstroemia speciosa (Lythraceae family) was prepared. H9c2 cells were used for treatment of Lagerstroemia speciosa extract with/without tumor necrosis factor (TNF). To examine NF-κB's activation, we performed an electrophoretic mobility shift assay (EMSA).

Results

The activation of NF-κB by TNF was completely blocked by a Lagerstroemia speciosa extract in a dose- and time-dependent manner in H9c2 cells.

Conclusion

Overall, our results indicate that Lagerstroemia speciosa can inhibit DNA-binding of NF-κB. This may explain its possible inhibition of diabetes-induced caridomyocyte hypertrophy.     

KIOM-79 prevents xylose-induced lens opacity and inhibits TGF-beta2 in human lens epithelial cells cultured under high glucose

Aim of the study

To investigate the effects of KIOM-79 in preventing the development of diabetic complications, such as cataracts.

Materials and methods

The inhibitory effects of KIOM-79 were assessed in a model of xylose-induced lens opacity and on changes mediated by high levels of glucose in human lens epithelial (HLE-B3) cells.

Results

In lenses treated with KIOM-79, opacity was significantly improved and glutathione (GSH) was increased compared to controls. In HLE-B3 cells treated with KIOM-79, high glucose-mediated increases in TGF-β2, αB-crystallin, and fibronectin were significantly inhibited in a dose-dependent manner. KIOM-79 decreased the phosphorylation of p-Smad2/3, pp38MAPK, pp44/42, and NF-κB signaling in cells grown under high glucose conditions.

Conclusion

KIOM-79 is protective against lens opacity and protects HLE-B3 cells from the toxic effects of high glucose. Therefore, KIOM-79 may provide a potential therapeutic approach for preventing diabetic complications, such as cataracts.     

The anti-inflammation effect of Moutan Cortex on advanced glycation end products-induced rat mesangial cells dysfunction and High-glucose–fat diet and streptozotocin-induced diabetic nephropathy rats

Ethnopharmacological relevance

Moutan Cortex (MC, family: Paeonia suffruticosa Andr.) is a well-known traditional herbal medicine that has been shown to hold a protective effect on inflammation in several diseases. However, its anti-inflammatory activity on diabetic nephropathy (DN) has been less reported. The present study was conducted to evaluate the potential attenuation activities of MC on inflammation in AGEs-induced rat mesangial cells dysfunction and high-glucose–fat diet and streptozotocin (STZ)-induced DN rats and explore the possible mechanism underlying its DN effect.

Materials and methods

The inflammation in mesangial cells (HBZY-1) was induced by 200 μg/ml advanced glycation end products (AGEs). DN rats model was established by an administration high-glucose–fat diet and an intraperitoneal injection of STZ (30 mg/kg). Interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) level in cell supernatant and rats serum were detected by appropriate kits. A co-culture system of mesangial cells and macrophages was performed to evaluate the migration of macrophages. Immunohistochemical assay was applied to examine transforming growth factor beta1 (TGF-β1), IL-6, MCP-1 and intercellular adhesion molecule-1 (ICAM-1) expression in kidney tissues of rats. Furthermore, western blot analysis was carried out to examine TGF-β1, IL-6, MCP-1, ICAM-1 and RAGE protein expressions in mesangial cells.

Results

Pretreatment with MC could significantly inhibit AGEs-induced migration of macrophages in the co-culture system of mesangial cell and macrophage. MC could decrease IL-6 and MCP-1 levels in serum of DN rats in a dose-dependent manner. Furthermore, MC also improved the blood glucose, serum creatinine and urine protein levels. Both immunocytochemistry analysis and western blot analysis showed that MC decreased significantly the over-expression of IL-6, MCP-1, TGF-β1, ICAM-1 and RAGE in mesangial cells or kidney tissues. Additionally, the protein expression of proinflammatory cytokine could also be down-regulated by the pretreatment of RAGE-Ab (5 μg/ml).

Conclusion

These findings indicated that the extract of MC had an amelioration activity on the inflammation in AGEs-induced mesangial cells dysfunction and high-glucose–fat diet and STZ-induced DN rats. The protective effect might be associated with the intervention of MC via target of RAGE. These findings suggested that MC might be a benefit agent for the prevention and treatment of DN.     

Aqueous extract of unripe Rubus coreanus fruit attenuates atherosclerosis by improving blood lipid profile and inhibiting NF-κB activation via phase II gene expression

Ethnopharmacological relevance

The fruit of Rubus coreanus has been used as a traditional herbal medicine for alleviation of inflammatory and vascular diseases in Asian countries.

Aim of the study

The anti-atherogenic effect of unripe Rubus coreanus fruit extract (URFE) and its underlying mechanism were analyzed in mice fed a high-fat diet (HFD) and in cell culture system.

Materials and methods

Mouse was freely given HFD alone or supplemented with URFE for 14 weeks, followed by analysis of atherosclerotic lesions and serum lipid levels. For in vitro assay, macrophages were pretreated with URFE, followed by stimulation with lipopolysaccharide (LPS). Expression levels of inflammatory genes (TNF-α, IL-1β, and iNOS) and phase II genes (heme oxygenase-1, glutamate cysteine lygase, and peroxiredoxine-1) as well as intracellular reactive oxygen species (ROS) level and NF-κB activation pathway were analyzed in cultured macrophages as well as mouse sera and aortic tissues.

Results

URFE supplementation reduced HFD-induced atherosclerotic lesion formation which was correlated with decreased levels of lipids, lipid peroxides, and inflammatory mediators (TNF-α, IL-1β, and nitric oxide) in sera as well as suppression of inflammatory gene in aortic tissues. In addition, pre-treatment of macrophages with URFE also suppressed LPS-induced NF-κB activation, ROS production, and inflammatory and phase II gene expressions. Inhibition of phase II enzyme and protein activities attenuated the suppressive effects URFE on ROS production, NF-κB activation, and inflammatory gene expression.

Conclusion

These results suggest that URFE attenuates atherosclerosis by improving blood lipid profile and inhibiting NF-κB activation via phase II antioxidant gene expression.     

Baicalein,an active component of Scutellaria baicalensis, protects against lipopolysaccharide-induced acute lung injury in rats

Ethnopharmacological relevance

Baicalein (BE), a phenolic flavonoid extracted mainly from the root of Scutellaria baicalensis Georgi, a Chinese herb, is traditionally used in oriental medicine. Several studies have demonstrated that BE exerts many beneficial effects including anti-inflammatory and antioxidant activities. However, its effect on acute lung injury (ALI) and the molecular mechanisms involved remain unclear and warrant further investigation. The aim of the study is to investigate whether BE improves lipopolysaccharide (LPS, intratracheally, i.t.)-induced ALI in rats, and further study the underlying mechanisms of its activity.

Material and methods

Rats were administrated with LPS (5 mg/kg/body weight, i.t.) through a 24-gauge catheter to establish the ALI model. The effects of BE on the levels of pro-inflammatory cytokines, nitrite/nitrate in bronchoalveolar lavage fluid, and the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and nuclear factor-kappa B (NF-κB) activation as well as the histopathological changes were evaluated.

Results

Results showed that BE (20 mg/kg, i.p.) treatment markedly attenuated LPS-induced lung edema, elevation of the levels of IL-1β, TNF-α, IL-6, CINC-3, and nitrite/nitrate in bronchoalveolar lavage fluid accompanied by a remarkable improvement of lung histopathological symptoms. The LPS-enhanced inflammatory cell infiltration and myeloperoxidase activity, O₂⁻ formation and the expression of inducible nitric oxide synthase and nitrotyrosin in lungs were all attenuated by BE. Notably, BE could augment Nrf2/HO-1 cascade, but inhibited NF-κB activation in LPS-instilled lungs that was strongly reversed by blocking HO-1 activity.

Conclusion

This study is the first to demonstrate that BE protects against LPS-induced ALI in rats. The underlying mechanisms may include inhibition of NF-κB-mediated inflammatory responses and upregulation of Nrf2/HO-1 pathway, which ultimately alleviates the pathological symptoms of ALI.     

Fuyuan Decoction inhibits nitric oxide production via inactivation of nuclear factor-κB in SW1353 chondrosarcoma cells

Ethnopharmacological relevance

Fuyuan Decoction (FYD) is an empirical formula of treating Bi Zheng in traditional Chinese medicine (TCM). Despite the fact that the efficiency of FYD on treating osteoarthritis has been verified in clinic, the underlying mechanisms are not totally understood. This study was to investigate the effects and mechanisms of FYD on nitric oxide (NO) production and nuclear factor (NF)-κB activation in interleukin (IL)-1β-stimulated chondrocytes.

Materials and methods

SW1353 human chondrosarcoma cells were pretreated with various concentrations of FYD-containing serum (FYD-CS), and then were stimulated by IL-1β. Amounts of NO were determined by Griess reaction assay. Inducible NO synthase (iNOS) expression, inhibitor-κBα (IκBα) degradation and nuclear translocation of p65 protein were determined by Western blot assay. DNA binding activity of NF-κB was determined by ELISA assay using Trans AM^™ kit for p65.

Results

10% and 20% (v/v) FYD-CS significantly decreased NO production in a concentration-dependent manner (p<0.05 or p<0.01) as compared to control in IL-1β-induced SW1353 cells. Besides, 10% and 20% FYD-CS also significantly reduced iNOS protein expression by about 60% and 70% (both p<0.01), respectively. Furthermore, 10% and 20% FYD-CS markedly decreased IκBα degradation by about 45% and 26% (p<0.01 or p<0.05), lessened P65 content in the nucleus by about 28% and 60% (both p<0.01), and repressed DNA binding activity of P65 by about 30% and 45% (both p<0.01) in IL-1β-induced SW1353 cells.

Conclusions

These findings suggested that FYD could inhibit NO production and iNOS expression in IL-1β-induced chondrocytes through suppressing NF-κB activation.     

Cardioprotective effects of the YiQiFuMai injection and isolated compounds on attenuating chronic heart failure via NF-κB inactivation and cytokine suppression

Ethnopharmacological relevance

The YiQiFuMai injection (YQFM) is a traditional Chinese medicine for the treatment of chronic heart failure (CHF). The present study not only evaluated the cardioprotective effect and anti-inflammatory mechanism of the YQFM injection in an experimental model of CHF but also investigated its bioactive constituents in vitro.

Materials and methods

The left anterior descending coronary artery (LAD) in rats was ligated to make an animal model of CHF. From this, electrocardiographic parameters and exterior signs of rat hearts were recorded. Additionally, the histopathology of heart tissues was examined, and parameters of inflammatory stress were measured. Experiments were performed over two months in LAD-ligation rats treated with YQFM or vehicle. Treatment with Captopril was used as a positive control, which has previously been shown to prevent CHF, and rats without LAD-ligation were used as a negative control. Furthermore, we screened and identified potential anti-inflammatory constituents by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) combined with NF-κB activity luciferase reporter assay systems. Further cytokine detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors from YQFM.

Results

The administration of YQFM significantly improved cardiac function and ameliorated the activity level of inflammatory mediators (such as tumor necrosis factor-alpha, interleukin-6, and interleukin-1β) in CHF rats. Eight potential anti-inflammatory ingredients, ginsenosides Rb1, Rg1, Rf, Rh1, Rc, Rb2, Ro, and Rg3, were characterized and confirmed. Among these compounds, ginsenoside Ro was revealed as a new NF-κB inhibitor.

Conclusion

The results suggested that NF-κB inactivation and cytokine suppression might be one of the main mechanisms of YQFM that caused ameliorative effects in CHF rats, and the major constituents of ginsenosides were identified playing a key role in the treatment of CHF.     

Inhibitory effects of salvianolic acid B on CCl4-induced hepatic fibrosis through regulating NF-κB/IκBα signaling

Ethnopharmacological relevance

Hepatic fibrosis, a precursor of liver cirrhosis, is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. Salvianolic acid B (SA-B) is one of water soluble compounds derived from Salvia miltiorrhiza Bunge (Danshen in Chinese) widely used for chronic liver diseases. In this study we investigated the protective effects of SA-B on CCl₄-induced hepatic fibrosis.

Materials and methods

Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl₄). Rats were divided into four groups, including normal controls (N group), model (M group), low SA-B of 10 mg/kg body weight (L group), or high SA-B of 20 mg/kg body weight (H group). After 6 weeks, macroscopic features of the liver and weight ratio of liver to body were measured. Liver fibrosis of the rats was evaluated by HE and Massion staining. Activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were checked with automated biochemistry analyzer. Serum levels of hyaluronic acid (HA), type IV collagen (IV-C), Laminin (LN) and procollagen III peptide (PIIIP) were detected by radioimmunoassay (RIA). The expression of NF-κB and IκBα was detected by western blotting.

Results

SA-B was shown to reduce CCl₄-induced hepatic fibrosis in rats. The serum levels of ALT, AST, and TBIL were significantly lower in the SA-B treatment groups than in the M group. Compared the M group, the serum levels of HA, LN, IV-C and PIIIP were decreased markedly after treatment with SA-B, especially in the H group. Treatment with SA-B at 10–20 mg/kg (L and N groups, respectively) dose-dependently decreased the expression of NF-κB in the nucleolus and increased the expression levels of NF-κB and IκBα protein in the cytoplasm compared to that of the M group.

Conclusions

This study reveals that SA-B could prevent the progression of liver angiogenesis and alleviate liver fibrosis possibly by regulating the expression of NF-κB and IκBα.     

KIOM-79 prevents S100b-induced TGF-β1 and fibronectin expression in mouse mesangial cells

Aim of the study

In this study, we investigated whether KIOM-79 inhibits transforming growth factor-beta 1 (TGF-β1) and fibronectin expression in mouse mesangial cells cultured under S100b, a specific ligand of the receptor for advanced glycation end products (RAGE).

Materials and methods

Cell counting kit (CCK-8) assay was employed to evaluate the viability of KIOM-79-treated mesangial cells. The effect of KIOM-79 on S100b-induced TGF-β1 and fibronectin expression was investigated using RT-PCR, ELISA, and Western blot on mesangial cells.

Results

KIOM-79 (up to 50 μg/ml) appeared to have no effect on cell viability. S100b induced an increase in the expression TGF-β1 and fibronectin. Expression of TGF-β1 and fibronectin was inhibited significantly by KIOM-79 treatment in mesangial cells. KIOM-79 also inhibited the expression of NF-kB and inactivated p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2 in mesangial cells. KIOM-79 pretreatment inhibited increased malondialdehyde (a product of lipid peroxidation and a marker for oxidative stress) levels in S100b-induced mesangial cells.

Conclusions

These data demonstrate that KIOM-79 inhibits expression of TGF-β1 and fibronectin through inactivation of MAPK/ERK1/2 signaling, reduction in malondiadehyde levels, and inhibition of NF-kB in mesangial cells cultured under diabetic conditions. KIOM-79 could be beneficial for preventing of the development of diabetic complications such as nephropathy.     

Eucommia ulmoides Oliv. bark. attenuates 6-hydroxydopamine-induced neuronal cell death through inhibition of oxidative stress in SH-SY5Y cells

Ethnopharmacological relevance

Eucommia ulmoides Oliv. Bark. (EUE) has commonly been used to fortify the muscles and lungs, lower blood pressure, prevent miscarriage, improve liver and kidney tone, and promote longevity as a traditional tonic medicine in Korea, China, and Japan.

Aim of the study

In this study, we investigated the mechanisms by which EUE protects neuronal cells from apoptosis induced by the Parkinson's disease (PD)-related neurotoxin, 6-hydroxydopamine (6-OHDA).

Materials and methods

We determined the neuroprotective effects of EUE on 6-OHDA-induced neuronal cell death, cytotoxicity, reactive oxygen species (ROS) production, and mitochondrial membrane dysfunction. Moreover, we examined whether EUE suppressed phosphorylation of c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3 beta (GSK-3β). Furthermore, the neuroprotective effects of EUE on 6-OHDA-induced activation of nuclear factor-kappa B (NF-κB) was studied in SH-SY5Y cells.

Results

Pretreatment of SH-SY5Y cells with EUE significantly reduced 6-OHDA-induced cell death and cytotoxicity. EUE inhibited 6-OHDA-induced generation of ROS, which conferred cytoprotection against 6-OHDA-induced oxidative injury. EUE treatment also strikingly inhibited 6-OHDA-induced mitochondrial dysfunction. In addition, EUE suppressed phosphorylation of JNK, PI3K/Akt, and GSK-3β. Furthermore, EUE blocked 6-OHDA-induced NF-κB nuclear translocation, an event downstream from JNK, PI3K/Akt, and GSK-3β phosphorylation. Moreover, chlorogenic acid (CGA), one of the active constituents of EUE, was also able to reduce 6-OHDA-induced toxicity in SH-SY5Y cells.

Conclusion

Taken together, these results suggest that EUE attenuates oxidative stress through activation of JNK, PI3K/Akt, GSK-3β, and NF-κB pathways, thereby protecting cells from neuronal cell death.