Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway / 中国中药杂志

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-β1(TGF-β1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-β1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Effect of Erjing Pills on alleviating neuroinflammation of AD rats based on TLR4/NF-κB/NLRP3 pathway and its mechanism / 中国中药杂志

This paper aimed to study the effect of Erjing Pills on the improvement of neuroinflammation of rats with Alzheimer's di-sease(AD) induced by the combination of D-galactose and Aβ_(25-35) and its mechanism. SD rats were randomly divided into a sham group, a model control group, a positive drug group(donepezil, 1 mg·kg~(-1)), an Erjing Pills high-dose group(9.0 g·kg~(-1)), and an Erjing Pills low-dose group(4.5 g·kg~(-1)), with 14 rats each group. To establish the rat model of AD, Erjing Pills were intragastrically administrated to rats for 5 weeks after 2 weeks of D-galactose injection. D-galactose was intraperitoneally injected into rats for 3 weeks, and then Aβ_(25-35) was injected into the bilateral hippocampus. The new object recognition test was used to evaluate the learning and memory ability of rats after 4 weeks of intragastric administration. Tissues were acquired 24 h after the last administration. The immunofluorescence method was used to detect the activation of microglia in the brain tissue of rats. The positive expressions of Aβ_(1-42) and phosphory protein Tau~(404)(p-Tau~(404)) in the CA1 area of the hippocampus were detected by immunohistochemistry. The levels of inflammatory factors interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in the brain tissue were determined by enzyme-linked immunosorbent assay(ELISA). Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptors 3(NLRP3) pathway-associated proteins in the brain tissue were determined by Western blot. The results showed that as compared with the sham group, the new object recognition index of rats in the model control group decreased significantly, the deposition of Aβ_(1-42) and p-Tau~(404) positive protein in the hippocampus increased significantly, and the levels of microglia activation increased significantly in the dentate gyrus. The levels of IL-1β, TNF-α, and IL-6 in the hippocampus of the model control group increased significantly, and the expression levels of TLR4, p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus increased significantly. Compared with the model control group, the Erjing Pill groups enhanced the new object recognition index of rats, decreased the deposition of Aβ_(1-42) and the expression of p-Tau~(404) positive protein in the hippocampus, inhibited the activation of microglia in the dentate gyrus, reduced the levels of inflammatory factors IL-1β, TNF-α, and IL-6 in the hippocampus, and down-regulated the expression levels of TLR4, p-NF-κB P65/NF-κB P65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus. In conclusion, Erjing Pills can improve the learning and memory ability of the rat model of AD presumably by improving the activation of microglia, reducing the expression levels of neuroinflammatory factors IL-1β, TNF-α, and IL-6, inhibiting the TLR4/NF-κB/NLRP3 neuroinflammation pathway, and decreasing hippocampal deposition of Aβ and expression of p-Tau, thereby restoring the hippocampal morphological structure.

Mechanism of Buyang Huanwu Decoction glycosides against atherosclerotic inflammation through NF-κB signaling pathway / 中国中药杂志

This study aims to explore the effect of Buyang Huanwu Decoction glycosides on the inflammatory response of apolipoprotein E~(-/-)(ApoE~(-/-)) mice and RAW264.7 cells through nuclear factor kappa-B(NF-κB) signaling pathway. In the in vivo experiment, ApoE~(-/-) mice were fed with high-fat diets for 12 weeks to induce the animal model of atherosclerosis, and 75 μg·mL~(-1) oxidized low-density lipoprotein(Ox-LDL) incubated RAW264.7 cells for 24 h to establish the atherosclerosis cell model. Automatic biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay(ELISA), Western blot, and droplet digital polymerase chain reaction(PCR) were used to determine the blood lipid levels, aortic intimal thickness, inflammatory factor content, NF-κB pathway-related proteins, and mRNA expression levels, and evaluate arterial atherosclerotic lesions and anti-atherosclerotic mechanisms of the drug. The model of atherosclerosis was successfully established in ApoE~(-/-) mice after 12 weeks of feeding with high-fat diets. In the model group, the plasma levels of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-C) were increased(P<0.01), the intima of the blood vessels was thickened, the levels of inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were increased, and the protein and mRNA expressions of NF-κB and inhibitor of NF-κB(IκBα) were significantly increased as compared with the control group. Compared with the model group, the high-dose Buyang Huanwu Decoction glycoside group decreased the plasma levels of TC, TG, and LDL-C, reduced the plaque area and thickness and the content of inflammatory factor TNF-α, and inhibited the protein and mRNA expressions of NF-κB and IκBα, with the effect same as Buyang Huanwu Decoction. In the in vivo experiment, 75 μg·mL~(-1) Ox-LDL stimulated RAW264.7 cells for 24 h to successfully establish a foam cell model. As compared with the control group, the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα in the model group increased. Compared with the model group, the middle-dose and high-dose Buyang Huanwu Decoction glycoside groups decreased the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα. The above results show that the glycosides are the main effective substances of Buyang Huanwu Decoction against atherosclerosis, which inhibit the NF-κB pathway and reduce the inflammatory response, thus playing the role against atherosclerotic inflammation same as Buyang Huanwu Decoction.

Hypoxia promotes lipopolysaccharide-induced CXCL10 expression in microglia / 生理学报

This study was aimed to investigate the effect of hypoxia on lipopolysaccharide (LPS)-induced CXC-chemokine ligand-10 (CXCL10) expression and the underlying mechanism. C57BL/6J mice were randomly divided into control, hypoxia, LPS, and hypoxia combined with LPS groups. The LPS group was intraperitoneally injected with 0.5 mg/kg LPS, and the hypoxia group was placed in a hypobaric hypoxia chamber (simulated altitude of 6 000 m). The serum and hippocampal tissue samples were collected after 6 h of the treatment. The levels of CXCL10 in the serum and hippocampal tissue of mice were detected by ELISA. The microglia cell line BV2 and primary microglia were stimulated with hypoxia (1% O2) and/or LPS (100 ng/mL) for 6 h. The mRNA expression level of CXCL10 and its content in culture supernatant were detected by real-time quantitative PCR and ELISA, respectively. The phosphorylation levels of nuclear factor κB (NF-κB) signaling pathway-related proteins, p65 and IκBα, were detected by Western blot. Moreover, after NF-κB signaling pathway being blocked with a small molecular compound, PDTC, CXCL10 mRNA expression level was detected in the BV2 cells. The results showed that in the LPS-induced mouse inflammatory model, hypoxia treatment could promote LPS-induced up-regulation of CXCL10 in both serum and hippocampus. Compared with the cells treated with LPS alone, the expression of CXCL10 mRNA and the content of CXCL10 in the culture supernatant of BV2 cells treated with hypoxia combined with LPS were significantly increased. The CXCL10 mRNA level of primary microglial cells treated with hypoxia combined with LPS was significantly up-regulated. Compared with the cells treated with hypoxia or LPS alone, the phosphorylation levels of p65 and IκBα in the BV2 cells treated with hypoxia combined with LPS were significantly increased. PDTC blocked the induction of CXCL10 gene expression by LPS in the BV2 cells. These results suggest that hypoxia promotes LPS-induced expression of CXCL10 in both animal and cell models, and NF-κB signaling pathway plays an important role in this process.

Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells / 南方医科大学学报

OBJECTIVE@#To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function.@*METHODS@#Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR.@*RESULTS@#Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05).@*CONCLUSION@#circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

Anti-inflammatory effects of Abelmoschus manihot (L.) Medik. on LPS-induced cystitis in mice: potential candidate for cystitis treatment based on classic use / 中国天然药物

Abelmoschus manihot (L.) Medik. (A. manihot) is a traditional Chinese herbal medicine with a variety of pharmacological properties. It was first recorded in Jiayou Materia Medica dating back to the Song dynasty to eliminate urinary tract irritation by clearing away heat and diuretic effect. However, its pharmacological action on urinary tract infections has not been investigated. The present study aims to evaluate the anti-inflammatory activity of A. manihot on a mouse model of lipopolysaccharide (LPS)-induced cystitis. The results showed that A. manihot decreased white blood cell (WBC) count in urine sediments of the cystitis mice, alleviated bladder congestion, edema, as well as histopathological damage, reduced the expression levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β simultaneously. Moreover, A. manihot administration significantly downregulated the expression levels of TLR4, MYD88, IκBα, p-IκBα, NF-κB p65, and p-NF-κB p65 in LPS-induced cystitis mice. These findings demonstrated the protective effect of A. manihot against LPS-induced cystitis, which is attributed to its anti-inflammatory profile by suppressing TLR4/MYD88/NF-κB pathways. Our results suggest that A. manihot could be a potential candidate for cystitis treatment.

Anti-inflammatory effects of Eucommia ulmoides Oliv. male flower extract on lipopolysaccharide-induced inflammation / 中华医学杂志(英文版)

BACKGROUND@#Eucommia ulmoides Oliv. is a medicinal plant native to China, with its bark (Eucommiae Cortex) traditionally being used for medicinal purposes. Previous research has shown that Eucommia male flowers can exert anti-inflammatory, analgesic, antibacterial, and other pharmacological effects, including immune regulation. This study explored the anti-inflammatory effects of the 70% ethanol extract of male flowers (EF) of E. ulmoides in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and LPS-administered mice.@*METHODS@#Cytotoxicity of EF for RAW 264.7 cells was investigated using Cell Counting Kit-8. The production of proinflammatory mediators, nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 was determined using enzyme-linked immunosorbent assays. IL-17, IL-23, and IL-10 mRNA levels were determined using quantitative real-time polymerase chain reaction. Activation of the nuclear factor (NF)-κB pathway in RAW 264.7 cells was investigated via Western blotting. In vivo anti-inflammatory effects of EF were studied in an LPS-induced acute inflammation mouse model by analyzing lung tissue histopathology, serum TNF-α and IL-6 levels, and myeloperoxidase (MPO) activity in lung tissue.@*RESULTS@#EF showed no significant cytotoxicity at concentrations from 10 to 60 μg/mL (cell viability > 80%) in the CCK-8 cell viability assay. EF inhibited the RAW 264.7 cell proliferation (EF 60 μg/mL, 120 μg/mL, and 250 μg/mL vs. negative control: 87.31 ± 2.39% vs. 100.00 ± 2.50%, P = 0.001; 79.01 ± 2.56 vs. 100.00 ± 2.50%, P < 0.001; and 64.83 ± 2.50 vs. 100.00 ± 2.50%, P < 0.001), suppressed NO (EF 20 μg/mL and 30 μg/mL vs. LPS only, 288.81 ± 38.01 vs. 447.68 ± 19.07 μmol/L, P = 0.004; and 158.80 ± 45.14 vs. 447.68 ± 19.07 μmol/L, P < 0.001), TNF-α (LPS+EF vs. LPS only, 210.20 ± 13.85 vs. 577.70 ± 5.35 pg/mL, P < 0.001), IL-1β (LPS+EF vs. LPS only, 193.30 ± 10.80 vs. 411.03 ± 42.28 pg/mL, P < 0.001), and IL-6 (LPS+EF vs. LPS only, 149.67 ± 11.60 vs. 524.80 ± 6.24 pg/mL, P < 0.001) secretion, and downregulated the mRNA expression of IL-17 (LPS+EF vs. LPS only, 0.23 ± 0.02 vs. 0.43 ± 0.12, P < 0.001), IL-23 (LPS+EF vs. LPS only, 0.29 ± 0.01 vs. 0.42 ± 0.06, P=0.002), and IL-10 (LPS+EF vs. LPS only, 0.30 ± 0.01 vs. 0.47 ± 0.01, P=0.008) in LPS-stimulated RAW 264.7 cells. EF inhibited the LPS-induced NF-κB p65 (LPS+EF 20 μg/mL and 30 μg/mL vs. LPS only: 0.78 ± 0.06 vs. 1.17 ± 0.08, P < 0.001; and 0.90 ± 0.06 vs. 1.17 ± 0.08, P =0.002) and inhibitor of kappa B (IκBα) phosphorylation (LPS+EF 20 μg/mL and 30 μg/mL vs. LPS only: 0.25 ± 0.01 vs. 0.63 ± 0.03, P < 0.001; and 0.31 ± 0.01 vs. 0.63 ± 0.03, P < 0.001), LPS+EF 30 μg/mL inhibited IκB kinase (IKKα/β) phosphorylation (LPS+EF 30 μg/mL vs. LPS only, 1.12 ± 0.14 vs. 1.71 ± 0.25, P = 0.002) in RAW 264.7 cells. Furthermore, EF 10 mg/kg and EF 20 mg/kg inhibited lung tissue inflammation in vivo and suppressed the serum TNF-α (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 199.99 ± 186.49 vs. 527.90 ± 263.93 pg/mL, P=0.001; and 260.56 ± 175.83 vs. 527.90 ± 263.93 pg/mL, P = 0.005), and IL-6 (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 41.26 ± 30.42 vs. 79.45 ± 14.16 pg/ ml, P = 0.011; and 42.01 ± 26.26 vs. 79.45 ± 14.16 pg/mL, P = 0.012) levels and MPO (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 3.19 ± 1.78 vs. 5.39 ± 1.51 U/g, P = 0.004; and 3.32 ± 1.57 vs. 5.39 ± 1.51 U/g, P = 0.006) activity in lung tissue.@*CONCLUSIONS@#EF could effectively inhibit the expression of inflammatory factors and overactivation of neutrophils. Further investigation is needed to evaluate its potential for anti-inflammation therapy.

Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model

Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

Huaiqihuang Granules () reduce proteinuria by enhancing nephrin expression and regulating necrosis factor κB signaling pathway in adriamycin-induced nephropathy / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Huaiqihuang Granules (, HQH), a mixture of Chinese herbs including Trametes robiniophila Murr, Fructus Lycii and Polygonatum sibiricum, on adriamycininduced nephropathy (ADRN) in rats and its underlying mechanisms.</p><p><b>METHODS</b>Rats with ADRN were divided into four groups: the sham group, the model group (distilled water), the low-dose HQH-treated (2 g/kg) group, and the high-dose HQH-treated (4 g/kg) group. Body weight and 24-h urinary protein (Upro) were checked every week. After 5-week intervention, at the end of the study, the rats were sacrificed and blood samples were collected for examination of biochemical parameters, including glomerular morphological makers, podocyte shape, cellular apoptosis, expressions of nephrin, inflammatory and apoptosis markers.</p><p><b>RESULTS</b>HQH ameliorated the rat's general status, proteinuria, renal morphological appearance and glomerulosclerosis. The decreased expression of nephrin in ADRN rats was increased by HQH, as well as the impaired podocyte foot process fusion. Cytosolic levels of p65 and inhibitor of nuclear factor κBα (IκBα) were decreased in ADRN rats, and recovered by the treatment of HQH. Consistently, the induced expression of tumor necrosis factor α (TNF-α), phosphorylated nuclear factor κB p65 (p-NFκB p65) and IκBα in ADRN were markedly suppressed by HQH. In addition, induction of Bax, cleaved caspase-3 and cytochrome C in ADRN rats were suppressed by HQH, indicating the amelioration of apoptosis.</p><p><b>CONCLUSION</b>HQH could ameliorate renal impairments in ADRN rats by increasing nephrin expression, inhibiting NF-κB signaling pathway via the down-regulation of p-NF-κB p65 and p-IκBα, and suppression of glomerular and tubular apoptosis.</p>

Interaction between polymorphisms of TLR4 gene G11367C in 3' untranslated region and IκB-α Hae III in acute pancreatitis and the degree of severity / 中南大学学报(医学版)

OBJECTIVE@#To investigate the interaction between polymorphism of Toll-like receptor 4 (TLR4) gene G11367C in 3' untranslated region (UTR) and inhibitor of nuclear factor kappaB (IκB)-α 
Hae III in acute pancreatitis (AP) and the degree of severity.
@*METHODS@#A total of 450 patients with confirmed AP (AP group), who came from the First Affiliated Hospital of Xinxiang Medical College from May 2013 to June 2015, were divided into a mild AP subgroup (MAP subgroup), a moderately severe AP (MSAP subgroup), and a severe acute AP (SAP subgroup) (n=150 in each group). One hundred fifty healthy persons were served as a control group. There was no significant difference in age, gender, ethnicity and birthplace among all groups. The genetic polymorphisms of TLR4 gene G11367C in 3' untranslated region and IκB-α Hae III were analyzed by polymerase chain reaction (PCR). Eligible participants were personally interviewed by a questionnaire. Unconditional logistic regression model and single factor analysis were performed to calculate the adjusted odds ratios (OR) and 95% confidence intervals (95% CI) of G11367C and IκB-α Hae III polymorphisms, respectively. The interaction of nucleotide polymorphisms was analyzed.
@*RESULTS@#The frequencies of G11367C (GC), IκB-α Hae III (AG) and IκB-α Hae III (GG) were 69.56%, 33.78% and 36.22% in the AP group; 49.33%, 24.67% and 26.00% in the MAP subgroup; 70.67%, 34.67% and 36.67% in the MSAP subgroup; 88.67%, 42.00% and 46.00% in the SAP subgroup and 26.67%, 14.00% and 14.67% in the control group, respectively. There was significant difference in the frequencies betweenc the AP group and the control group, or among each AP subgroup (all P1). Similarly, there were also positive interactions in the pathogenesis of AP between G11367C (GC) and IκB-α Hae III (AG) (All γ>1). 
@*CONCLUSION@#These carriers of G11367C(GC), IκB-α Hae III(AG) and IκB-α Hae III (GG) genotypes may have a high risk of AP occurency, and there are significant interactions between genetic polymorphisms of G11367C and IκB-α Hae III, which increaes the risk of the occurrence and development of AP.

Hydroxysafflor yellow A attenuate lipopolysaccharide-induced endothelium inflammatory injury / 中国结合医学杂志

<p><b>OBJECTIVE</b>This study observed attenuating effect of hydroxysafflor yellow A (HSYA), an effective ingredient of aqueous extract of Carthamus tinctorius L, on lipopolysaccharide (LPS)-induced endothelium inflammatory injury.</p><p><b>METHODS</b>Eahy926 human endothelium cell (EC) line was used; thiazolyl blue tetrazolium bromide (MTT) test was assayed to observe the viability of EC; Luciferase reporter gene assay was applied to measure nuclear factor-κB (NF-κB) p65 subunit nuclear binding activity in EC; Western blot technology was used to monitor mitogen activated protein kinase (MAPKs) and NF-κB activation. Reverse transcription polymerase chain reaction (RT-PCR) method was applied to observe intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin mRNA level; EC surface ICAM-1 expression was measured with flow cytometry and leukocyte adhesion to EC was assayed with Rose Bengal spectrophotometry technology.</p><p><b>RESULTS</b>HSYA protected EC viability against LPS-induced injury (P <0.05). LPS-induced NF-κB p65 subunit DNA binding (P <0.01) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) phosphorylation was inhibited by HSYA. HSYA attenuated LPS triggered ICAM-1 and E-selectin mRNA levels elevation and phosphorylation of p38 MAPK or c-Jun N-terminal kinase MAPK. HSYA also inhibited LPS-induced cell surface ICAM-1 protein expression P <0.01) and leukocyte adhesion to EC (P <0.05).</p><p><b>CONCLUSION</b>HSYA is effective to protect LPS-induced high expression of endothelium adhesive molecule and inflammatory signal transduction.</p>

Mechanism of colon cancer cell apoptosis induced by telocinobufagin: role of oxidative stress and apoptosis pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis.</p><p><b>METHODS</b>MTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting.</p><p><b>RESULTS</b>Telocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells.</p><p><b>CONCLUSION</b>Telocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.</p>

The antagonistic effect and mechanism of N-acetylcysteine on acrylamide-induced hepatic and renal toxicity / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>The aim of this study is to investigate hepatic and renal toxicity of acrylamide (ACR) , the antagonistic effect and possible mechanism of N-acetylcysteine (NAC) on the toxicity.</p><p><b>METHODS</b>Forty female SD rats were randomly divided into four groups. All the rats were administrated by intraperitoneal(i.p.) injection and 1.5 hours later by gavage. The control group was administrated with 0.9% NaCl by i.p. injection and gavaged with 0.9% NaCl. The NAC group was administrated with 200 mg/kg NAC by injection and gavaged with 0.9% NaCl. The ACR group was administrated with 0.9% NaCl by injection and gavaged with 40 mg/kg ACR. The combined treatment group was administrated with 200 mg/kg NAC by i.p. injection and gavaged with 40 mg/kg ACR. The rats were administrated once a day for 2 weeks. After 24 hours of the last administration, the rats were decapitated. The blood was collected, the liver and kidney were separated. The body weight, organ coefficient and serum biochemical parameters were measured, and the pathological changes of the tissues were examined with a microscope. Then the expression of NF-κB p65, IκB-α and COX-2 were detected by Western blot.</p><p><b>RESULTS</b>From the second day to the end of the exposure, the body weight of rats in the ACR group was statistically lower than that in the control group (P<0.05) . Compared with the combined treatment group, the body weight in the ACR group statistically decreased in the second and third days (P < 0.05) . The liver and kidney organ coefficients in the ACR group were (4.159%±.371%) and (0.764%±0.068%) respectively, which increased statistically when compared with the control group (P < 0.05) . The contents of ALT, AST and Cr in the serum in the ACR group were (77.370±16.397) U/L、(379.410±57.817) U/L and (77.812±6.391) μmol/L respectively, which were not significantly different with those in the control group and the combined treatment group (P>0.05) . The content of BUN in the serum in the ACR group was (7.005±1.009) mmol/L, which was statistically higher than that in the control group (P<0.05) . Histopathology results showed unclear boundary and nucleus pyknosis in hepatocytes, loose and disordered structures of hepatic cords in the ACR group, but no obvious pathology changes were observed in the kidneys of each group. In the Western blot results, the expression of nuclear NF-κB p65 and COX-2 in the liver in the ACR group was statistically higher than that in the control group and the combined treatment group (P<0.05) , and the expression of IκB-α in the liver in the ACR group statistically decreased compared with the control group and the combined treatment group (P<0.05) . The expression of total NF-κB p65 in the liver in the ACR group was statistically higher than that in the control group (P<0.05) .</p><p><b>CONCLUSION</b>Under the conditions of this experiment, ACR may induce hepatic toxicity through the activation of NF-κB signaling pathway, and NAC could antagonize the hepatic toxicity of ACR by inhibiting the NF-κB signaling pathway, whereas the toxic effect of ACR on kidney needs to be further studied.</p>

Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR / 药学学报

This study aims to investigate the function of two SNPs (rs8904C > T and rs696G >A) in 3' untranslated region (3'UTR) of NFKBIA gene by constructing luciferase reporter gene. A patient's genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3'UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696G, pGL3-rs8904C/rs696A, pGL3-rs8904T/rs696G and pGL3-rs8904T/rs696A. Then these plasmids were transfected into LS174T cells and the luciferase activity was detected. Compared with pGL3-vector transfected cells (negative control), the luciferase activity of the four kinds of recombinant plasmids was significantly decreased (P < 0.001). For rs696G > A, the luciferase activity of the recombinant plasmids containing A allele (pGL3-rs8904C/rs696A and pGL3-rs8904T/rs696A) was about 45.1% (P < 0.05) and 56.1% (P < 0.001) lower than those containing G allele (pGL3-rs8904C/rs696G and pGL3-rs8904T/rs696G), respectively. For rs8904C > T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR were constructed successfully and rs696G > A could decrease the luciferase activity while rs8904C >T didn't have much effect on the luciferase activity.

Establishment and Evaluation of Hypertensive Rat Model with Excessive Accumulation of Phlegm-dampness Syndrome / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe mainfestations of syndrome and biochemical indices of hypertensive model rats with excessive accumulation of phlegm-dampness syndrome (EAPDS), and to explore its possible pathological mechanism.</p><p><b>METHODS</b>EAPDS rat model was prepared in 50 Wistar rats by feeding with high fat forage. Meanwhile, a normal control group consisting of 10 Wistar rats was set up by feeding with normal forage. After 25-week continuous feeding, 22 rats with body weight (BW) and blood pressure (BP) exceeding 25% those of the control group were selected as a model group. BW, BP, blood lipids, and related serological indicators were detected in all rats. Morphological changes of target organs were observed. mRNA expression levels of leptin receptor (LepR), Janus kinase2 (Jak2), signal transducer and activator of transcription 3 (Stat3), suppressor of cytokine signaling-3 (Socs3), angiotensin II receptor type 1 (AT1), angiotensin II receptor type 2 (AT2), phosphatidylinositol 3 kinase (P13K), serine threonine kinase (Akt), nuclear factor of kappa B (NF-κBp65), inhibitor of nuclear factor kappa-B kinase α (IKKα), NF-kappa-B inhibitor β (lKKβ), NF-kappa-B inhibitor α (IKBα), and AMP-activated protein kinase (AMPK) were detected by quantitative real-time PCR (qPCR). Expression levels of AT1 and LepR in aorta were detected by immunohistochemical assay and Western blot respectively.</p><p><b>RESULTS</b>Compared with the control group, BW, BP, and blood lipids increased; serum levels of leptin (Lep) , Ang II, Hcy, ET-1, TNF-α, IL-6, and p2-MG increased, but NO decreased in the model group (P < 0.05, P < 0.01). Aortal endothelial injury and smooth muscle cell proliferation occurred in the model group, accompanied with heart and renal injury. Compared with the control group, mRNA expression levels of LepR, Jak2, Stat3, Socs3, AT1 , PI3K, Akt, NF-κB p65, IKKβ, IKBα, and AMPK in aorta were up-regulated significantly (P < 0.05), while the expression of IKKa decreased (P < 0.05). Immunohistochem- ical staining showed, brownish yellow deposit of AT1 and LepR was obviously increased, with more extensively positive distribution. Western blot results showed, as compared with the control group, protein expression levels of AT1 and LepR obviously increased in the model group (P < 0.05).</p><p><b>CONCLUSIONS</b>Model rats exhibited typical syndromes of EAPDS. They put up weight with fat abdomen, gloomy hair, poor appetite, hypersomnia, lowered activities , reduced food intake, loose stool, dark red tongue, white tongue with white, thick, greasy fur. Lep could be taken as one of objective indicators for evaluating hypertension rat model with EAPDS.</p>

Preventive effects of the polysaccharide of Larimichthys crocea swim bladder on carbon tetrachloride (CCl4)-induced hepatic damage / 中国天然药物

The aim of the present study was to determine the preventive effects of the polysaccharide of Larimichthys crocea swim bladder (PLCSB) on CCl4-induced hepatic damage in ICR mice. The in vitro preventive effects of PLCSB on CCl4-induced liver cytotoxic effect were evaluated in BRL 3A rat liver cells using the MTT assay. The serum levels of AST, ALT, and LDH in mice were determined using commercially available kits. The levels of IL-6, IL-12, TNF-α, and IFN-γ were determined using ELISA kits. The pathological analysis of hepatic tissues was performed with H and E staining, and the gene and protein expressions were determined by RT-PCR and Western blotting, respectively. PLCSB (20 μg·mL(-1)) could increase the growth of BRL 3A rat liver cells treated with CCl4. The serum levels of AST, ALT, and LDH were significantly decreased when the mice were treated with two doses of PLCSB, compared with the control mice (P < 0.05). PLCSB-treated groups also showed reduced levels of the serum pro-inflammatory cytokines IL-6, IL-12, TNF-α, and IFN-γ. PLCSB could decrease the liver weight, compared to the CCl4-treated control mice. The histopathology sections of liver tissues in the 100 mg·kg(-1) PLCSB group indicated that the animals were recovered well from CCl4 damage, but the 50 mg·kg(-1) PLCSB group showed necrosis to a more serious extent. The 100 mg·kg(-1) PLCSB group showed significantly decreased mRNA and protein expression levels of NF-κB, iNOS, and COX-2, and increased expression of IκB-α compared with the CCl4-treated control group. In conclusion, PLCSB prevented from CCl4-induced hepatic damage in vivo.

Ursolic acid inhibits corneal graft rejection following orthotopic allograft transplantation in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of ursolic acid on corneal graft rejection in a rat model of othotopic corneal allograft transplantation.</p><p><b>METHODS</b>Forty-eight recipient Wistar rats were divided into normal control group with saline treatment (group A), autograft group with saline treatment (group B), SD rat allograft group with saline treatment (group C), and SD rat allograft group with intraperitoneal ursolic acid (UA) treatment group (group D). The rats received saline or UC (20 mg·kg(-1)·d(-1)) treatment for 12 days following othotopic graft transplantation. The grafts were evaluated using the Larkin corneal rejection rating system, and the graft survival was assessed with Kaplan-Meier analysis. On day 14, the grafts were harvested for histological examination, Western blotting, and assessment of expressions of interlukin-2 (IL-2), interferon-γ (IFN-γ), nuclear transcription factor-κB (NF-κB) p65, vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1).</p><p><b>RESULTS</b>The allograft survival was significantly longer in group D than in group C (29.12±9.58 vs 9.67±2.16 days, P<0.05). UC treatment obviously reduced the expression levels of IL-2, IFN-γ, NF-κBp65, ICAM-1 and VEGF and increased inhibitory kappa B alpha (IκB-α) expression in the grafts, where no obvious inflammatory cell infiltration or corneal neovascularization was found.</p><p><b>CONCLUSION</b>As a NF-κB inhibitor, ursolic acid can prevent corneal neovascularization and corneal allograft rejection to promote graft survival in rats following orthotopic corneal allograft transplantation.</p>

Qinghuachang Decoction Inhibited NF-kappaB Activation in LPS-induced Human Enterocytes / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore anti-inflammation and mechanism of Qinghuachang Decoction (QD) by using LPS stimulated differentiated colon cancer Caco-2 cells (as an inflammation model of human enterocytes).</p><p><b>METHODS</b>QD was prepared. Human colonic epithelial Caco-2 cells were cultured. Expressions of TNF-alpha and IL-8 were determined using ELISA. Expressions of inhibitory Kaba protein (IkappaB-alpha), phosphorylated inhibitory Kaba protein (p-lkappaB-alpha), nuclear transcription factor p50 (p50), and nuclear transcription factor ReIA (ReIA) protein were determined by Western blot.</p><p><b>RESULTS</b>Compared with the negative control group (without LPS stimulation), LPS stimulated the release of IL-8 and TNF-alpha in Caco-2 cells (P < 0.05). QD treatment could reduce the secretion of TNF-alpha and IL-8 induced by LPS in a dose dependent manner (P < 0.05). QD at 0, 5, 10, and 50 microg/mL had no significant effect on Caco-2 cell survival rates (P > 0.05), with no statistical difference among various concentrations (P > 0.05). QD could significantly suppress nuclear factor-kappa B (NF-kappaB) phosphorylation stimulated by LPS. The expression of p-IKappaB-alpha was decreased with increasing concentrations of QD (P < 0.05). There was no obvious change in IKB-alphaB expressions (P > 0.05). Expressions of p50 and ReIA decreased with increasing concentrations of QD (P < 0.05). Both of them were in a dose dependent manner.</p><p><b>CONCLUSION</b>QD inhibited LPS mediated NF-kappaB activation, which might be one of its mechanisms for treating inflammatory bowel disease (IBD).</p>

Nuclear factor-κB pathway mediates the effects of CD137 signaling on NFATc1 expression in mice vascular smooth muscle cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe whether CD137 signaling could affect the nuclear factor of activated T cells c1 (NFATc1) expression through nuclear factor-κB (NF-κB) pathway in mice aortic vascular smooth muscle cells (VSMCs).</p><p><b>METHODS</b>Adherence methods for tissues explants were used for primary culture of mouse aortic VSMCs. The mRNA expression of CD137 and NFATc1 was detected by real-time quantitative PCR (RT-qPCR). The VSMCs protein expression of IκB-α, NF-κB p65, phospo-p65 and NFATc1 was determined by Western blot. The level of CD137 was measured by Flow Cytometry (FCM).</p><p><b>RESULTS</b>(1) The mRNA and protein expression of CD137 in VSMCs was significantly upregulated at 24 h after co-culture with TNF-α (10 ng/ml, all P < 0.05). (2) Compared with the control group, the level of p-NF-κB p65 in cytoplasm and nucleus was significantly increased (8.34 ± 0.28 vs. 1, P < 0.05, and 2.64 ± 0.42 vs. 1, P < 0.05) while the level of IκB-α was reduced (1 vs. 2.70 ± 0.28, P < 0.05) after co-treatment with agonist-CD137 mAb, above effects were partly blocked by adding specific NF-κB inhibitor PDTC (30 µmol/L: 1.15 ± 0.14 vs. 8.34 ± 0.28, P < 0.05, and 2.09 ± 0.12 vs. 2.64 ± 0.42, P < 0.05, and 1.78 ± 0.74 vs. 1, P < 0.05). (3) The mRNA (2.07 ± 0.09 vs. 1, P < 0.05) and protein (1.75 ± 0.07 vs. 1, P < 0.05) expression of NFATc1 was significantly upregulated by agonist CD137mAb compared with the control group, and these effects could be reversed by PDTC (1.15 ± 0.07 vs. 2.07 ± 0.09, P < 0.05, and 0.90 ± 0.11 vs. 1.75 ± 0.07, P < 0.05).</p><p><b>CONCLUSION</b>CD137 signaling could affect the NFATc1expression in VSMCs through NF-kappaB pathway.</p>

Inhibitory effect of kaempferol on inflammatory response of lipopolysaccharide-stimulated human mast cells / 药学学报

This study is to investigate the inhibitory effect of kaempferol on inflammatory response of lipopolysaccharide(LPS)-stimulated HMC-1 mast cells. The cytotoxicity of kaempferol to HMC-1 mast cells were analyzed by using MTT assay and then the administration concentrations of kaempferol were established. Histamine, IL-6, IL-8, IL-1β and TNF-α were measured using ELISA assay in activated HMC-1 mast cells after incubation with various concentrations of kaempferol (10, 20 and 40 µmol.L-1). Western blot was used to test the protein expression of p-IKKβ, IκBα, p-IκBα and nucleus NF-κB of LPS-induced HMC-1 mast cells after incubation with different concentrations of kaempferol. The optimal concentrations of kaempferol were defined as the range from 5 µmol.L-1 to 40 µmol.L-1. Kaempferol significantly decreased the release of histamine, IL-6, IL-8, IL-1β and TNF-α of activated HMC-1 mast cells (P<0.01). After incubation with kaempferol, the protein expression of p-IKKβ, p-IKBa and nucleus NF-κB (p65) markedly reduced in LPS-stimulated HMC-1 mast cells (P<0.01). Taken together, we concluded that kaempferol markedly inhibit mast cell-mediated inflammatory response. At the same time, kaempferol can inhibit the activation of IKKβ, block the phosphorylation of IκBα, prevent NF-KB entering into the nucleus, and then decrease the release of inflammatory mediators.