Biancaea decapetala (Roth) O.Deg. extract exerts an anti-inflammatory effect by regulating the TNF/Akt/NF-κB pathway.

BACKGROUND: Biancaea decapetala (Roth) O.Deg. (Fabaceae) is used to treat colds, fever, and rheumatic pain caused by inflammation. However, the mechanism underlying its anti-inflammatory properties remains unclear. PURPOSE: This study aimed to evaluate the anti-inflammatory activity of Biancaea decapetala extract (BDE) in vitro and in vivo and explore the possible underlying mechanism and potential targets. METHODS: The release of nitric oxide (NO) and inflammatory cytokines in LPS-stimulated RAW264.7 cells and rats were measured using Griess reagent and enzyme-linked immunosorbent assay (ELISA). Hematoxylin and eosin (H&E) staining was employed to examine the pathology of animal tissues. Transcriptome analysis was performed to screen the pathways related to BDE-mediated inhibition of inflammation, and the expression of related proteins was measured using real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, ELISA, and immunofluorescence methods. Surface Plasmon Resonance (SPR) and the Drug Affinity Reaction Target Stability (DARTS) method were used to verify whether BDE binds to TNF-α target protein, while a L929 cell model and NF-κB gene reporter systematic method were used to investigate the inhibitory effect of BDE on the activity of TNF-α protein. RESULTS: BDE inhibited the expression of TNF-α, IL-1ß, IL-6, and NO in RAW264.7 cells and rats, and improved the pathological changes in lung tissue. RNA-seq showed that BDE may regulate the TNF/Akt/NF-κB pathway to inhibit inflammation onset. BDE significantly downregulated the mRNA expression of TNF-α, IL-6, IL-1ß, and that of relevant proteins, including TNF-α, p-p65, p-Akt, p-IκBα. Furthermore, BDE inhibited the nuclear translocation of NF-κB (p65) and the activation of the Akt pathway by SC79. The L929 cell model, luciferase reporter gene analysis, DARTS, and SPR experiments showed that BDE may bind to TNF-α and inhibit the TNF-α-NF-κB pathway. CONCLUSION: BDE may target TNF-α to inhibit the TNF/Akt/NF-κB pathway, thereby attenuating inflammation. These findings reveal the anti-inflammatory effects and mechanisms of BDE and provide a theoretical basis for the further development and utilization of BDE.

Quantification of six volatile oil constituents of Oleum Cinnamomi in rat plasma and multiple tissues using GC-MS and its application to pharmacokinetic and tissue distribution studies.

Oleum Cinnamomi is the essential oil obtained from the herb Fructus Cinnamomi which is used by the Hmong people in traditional medicine for the treatment of various diseases. At present, there are a variety of marketed preparations with it as the main medicine on the market. Information regarding the in vivo process of it is lacking, which has become a bottleneck restricting its development and utilization. In view of this, a GC-MS SIM analysis method was established for the simultaneous determination of six main volatile components [eucalyptol, p-cymene, 4-carvomenthenol, 4-isopropyl-2-cyclohexenone, α-terpineol, and 2-(4-Methylphenyl)-propan-2-ol] in plasma and ten tissues of rats to study their pharmacokinetic and distribution characteristics in vivo. The pharmacokinetic results showed that the t1/2 of each index was 0.41-1.66 h, Tmax was 0.16-0.68 h, Cmax was 13.66-2015.02 ng/mL, AUC0-t was 12.84-4299.00 h·ng/mL, CLZ/F was 1750.93-107013.11 mL/h/kg. This meant that the six components could be absorbed quickly, had a short residence time, and be eliminated quickly in the body. Among them, eucalyptol has the highest degree of absorption and a larger amount of entering the body. Moreover, the Cmax and AUC0-t of the six components increased correspondingly with the increase of the dose, indicating that the concentration of Oleum Cinnamomi in the rat plasma was dose-dependent. At different time points, the six components were widely distributed with uneven characteristics in the body. The six components mainly tend to be distributed in stomach, small intestine, and liver, followed by kidney, spleen, heart, and brain, and to a lesser extent in lung, skin, and muscle. And the six components were eliminated quickly in each tissue. The pharmacokinetic process and tissue distribution characteristics of Oleum Cinnamomi were expounded in this study, which can provide scientific theory for the in-depth development and guidance of clinical drug use of Oleum Cinnamomi, and at the same time provide a medicinal material basis for the in-depth development and utilization of Oleum Cinnamomi.

[Uptake and transport of Laportea bulbifera extract in Caco-2 cell model].

Laportea bulbifera extract is effective in resisting inflammation and shows a good therapeutic effect on rheumatoid arthritis in rats. However, the absorption characteristics of active components in L. bulbifera extract in Caco-2 cells are still unclear, which limits the in-depth development of L. bulbifera resources. The purpose of this study was to investigate the absorption and transport mechanism of the active components of L. bulbifera extract in the Caco-2 cell model and explore the effects of different factors(concentration, time, pH value, temperature, and efflux transporter inhibitor) on its uptake and transport. The results showed that L. bulbifera extract at the concentration of 2.0-8.0 mg·mL~(-1) showed no toxicity to Caco-2 cells. The uptake and transport of L. bulbifera extract in the Caco-2 cell model were concentration-dependent and time-dependent. The main absorption mechanism was passive diffusion, and acidic condition(pH 5.0-6.0) and 37 ℃ were more favorable for drug absorption. P_(app)>1.0×10~(-6 )cm·s~(-1) of each component indicated that L. bulbifera was a moderately absorbed drug. P-gp, MRP2, and BCRP were not involved in its uptake and transport.

[Potential pharmacodynamic substances of Laportea bulbifera in treatment of rheumatoid arthritis based on serum pharmacochemistry and pharmacology].

The present study investigated the pharmacodynamic material basis of Laportea bulbifera in the treatment of rheumatoid arthritis. Firstly, human rheumatoid arthritis fibroblast-like synoviocyte line MH7A was cultured in vitro and treated with tumor necrosis factor alpha(TNF-α, 50 ng·mL~(-1)). The proliferation and the levels of inflammatory cytokines such as prostaglandin E2(PGE2), interleukin-1ß(IL-1ß), and interleukin-6(IL-6) of the MH7A cells exposed to the serum containing L. bulbifera were determined to evaluate the anti-rheumatoid arthritis effects of the serum. Furthermore, the ultra-performance liquid chromatography tandem mass spectrometry fingerprints of the L. bulbifera crude extract, the drug-containing serum, and the drug-free serum were compared to identify the compounds newly generated in the serum after oral administration of the extract. According to the peak areas of common peaks and the results of anti-rheumatoid arthritis effect test, the active components were identified. The serum containing L. bulbifera significantly inhibited the proliferation of the MH7A cells activated by TNF-α and the expression of PGE2, IL-6, and IL-1ß. Thirty newly generated compounds were detected in the drug-containing serum. Among them, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, rutin, isoquercitrin, luteoloside, kaempferol-3-O-rutinoside, and quercitrin were also present in the crude extract. Twelve characteristic peaks(3, 7, 8, 14, 18, 19, 21, 23, 24, m6, m7, and m15) were significantly correlated with the pharmaceutical effect. According to the correlations, neochlorogenic acid, cryptochlorogenic acid, and chlorogenic acid had great contributions to the anti-rheumatoid arthritis activity. This study preliminarily clarified the potential pharmacodynamic substances of L. bulbifera in the treatment of rheumatoid arthritis, which laid a theoretical and experimental foundation for further development and application of the medicinal plant.

[Establishment of PK-PD model in anti-inflammatory active components in Inula cappa extract based on lipopolysaccharide-induced in vitro inflammation model].

In the present study, a pharmacokinetics(PK)-pharmacodynamics(PD) model in the anti-inflammatory active components in Inula cappa extract was established based on the lipopolysaccharide(LPS)-induced in vitro inflammation model in order to clarify the relationship between the dynamic changes of anti-inflammatory active components in inflammatory cells and their efficacy. Firstly, the inflammation model in vitro was induced by 1 µg·mL~(-1) LPS in RAW264.7 cells for 24 h. After treatment with 400 µg·mL~(-1) I. cappa extract, the pharmacokinetics(PK) of five anti-inflammatory active components, including luteolin(LUT), chlorogenic acid(CA), cryptochlorogenic acid(CCA), 3,4-dicaffeoylquinic acid(3,4-DCQA), and 4,5-dicaffeoylquinic acid(4,5-DCQA), in normal cells and inflammatory cells was compared. Meanwhile, the PD study was carried out by measuring the inflammatory factors NO and TNF-α in the cell supernatant at each time point, which was fitted with PK by the Phoenix Model in the WinNonlin 8.2 to establish the PK-PD model for five components including LUT, CA, CCA, 3,4-DCQA, and 4,5-DCQA. The results showed that compared with normal cells, the model cells showed increased or decreased uptake of five components, advanced T_(max), faster absorption, prolonged MRT and t_(1/2), and increasing or decreasing trend of CL_(z/F) and V_(z/F). When NO was used as the efficacy index, the PK-PD model after the integration of the multi-effect components in I. cappa was E=7.45×\[1-Ce~(5.74)/(78.24~(5.74)+Ce~(5.74))\], while with TNF-α as the efficacy index, the PK-PD model after the integration of the multi-effect components in I. cappa was E=79.28×[1-Ce~(6.45)/(85.10~(6.45)+Ce~(6.45))]. The results of the study suggested that the inflammatory state could change the cellular PK of I. cappa. The anti-inflammatory effect of active components in I. cappa might be related to the down-regulation of the secretion of NO and TNF-α in inflammatory cells, and NO and TNF-α might serve as the anti-inflammatory targets of active components of I. cappa.

Epidermal Growth Factor Receptor Gene Mutation Status in Primary Lung Adenocarcinoma and Corresponding Bone Metastases.

BACKGROUND: The aim of this study was to compare epidermal growth factor receptor (EGFR) mutations between primary tumors and corresponding bone metastases (BMs) in lung adenocarcinoma. MATERIALS AND METHODS: In total, 115 paired primary lung adenocarcinoma and corresponding BM tumors were analyzed for EGFR mutations by Amplification Refractory Mutation System. RESULTS: EGFR mutations were detected in 61 primary lung adenocarcinomas (53.04%) and in 67 corresponding metastases (58.26%), respectively. The EGFR mutation rate was significantly higher in female and in never-smoker patients. The consistency of EGFR mutations between the 115 matched BMs and primary tumor tissue samples reached to 80.87%, and the disparity was 19.13%. Mutations in exons 19 (19-del) and 21 (point mutation L858R) were the predominant mutation type. CONCLUSIONS: The concordance rate demonstrated the feasibility of EGFR mutations in corresponding metastases using Amplification Refractory Mutation System when the primary tumor tissue is unavailable in the lung adenocarcinoma patients, and the inconsistency indicates that corresponding metastasis being screened simultaneously with the primary tumor samples may present some supplementary information for the patients.

Ursodeoxycholic acid protects interstitial Cajal-like cells in the gallbladder from undergoing apoptosis by inhibiting TNF-α expression.

Hypomotility is a common symptom of gallstone disease, which is accompanied by a loss of interstitial Cajal-like cells (ICLCs) in the gallbladder. Ursodeoxycholic acid (UDCA) is widely used in treating gallstone disease, and has shown anti-apoptotic and anti-inflammatory effects apart from its ability to dissolve gallstones. In this study, we investigated the anti-apoptotic and anti-inflammatory effects of UDCA on ICLCs in guinea pigs with gallstones. Guinea pigs were fed a high-cholesterol diet for 8 weeks to induce the formation of gallstones. A group of animals was administered UDCA (50 mg·kg-1·d-1, ig) simultaneously. At the end of 8 weeks, the animals were euthanized with anesthesia, cholecystectomy was performed immediately and gallbladder was collected for further analysis. We showed that in the model group the contractility of gallbladder muscle strips in response to both acetylcholine (ACh) and CCK-8 was severely impaired, which was significantly improved by UDCA administration. Furthermore, UDCA administration significantly reduced the apoptotic ratio of ICLCs, based on the observation of co-localization imaging of apoptotic cells and c-kit-positive cells. Western blotting analysis and real-time PCR results revealed that the TNF-α/Caspase8/Caspase3 pathway was suppressed in the UDCA-treated animals, confirming the anti-apoptotic effect of UDCA in the gallbladder. The H&E staining showed that UDCA administration significantly attenuated inflammatory cell infiltration in the gallbladder wall. In conclusion, UDCA can protect ICLCs in the gallbladder from undergoing apoptosis by inhibiting the TNF-α/Caspase8/caspase3 pathway.

[Protective effect of Polygonum orientale flower extract on H2O2-induced oxidative damage of HUVEC cells].

To investigate the protective effects and mechanism of Polygonum orientale flower extract on H2O2-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H2O2 was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 µmol·L⁻¹ H2O2 for 0.5 h. Treatment of H2O2 also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H2O2 model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H2O2-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.

[Chemical constituents from the fruits of Vitex negundo var. cannabifolia and their biological activities in vitro].

Chemical constituents from the fruits of Vitex negundo var. cannabifolia and their nitric oxide (NO) inhibitory and cytotoxic activities were investigated. The compounds were isolated and purified by various column chromatography, and their structures were identified by physiochemical properties and spectroscopic data. Thirteen lignans and six phenolic compounds were isolated from the CH2Cl2 extract of the fruits of V. negundo var. cannabifolia, respectively. Their structures were elucidated as 6-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde (1), vitedoin A (2), vitexdoin F (3), detetrahydroconidendrin (4), vitexdoin E (5), 4-oxosesamin (6), L-sesamin (7), (+)-beechenol (8), ligballinol (9), 2-(4-hydroxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (10), (-)-pinoresinol (11), balanophonin (12), thero-guaiacylglycerol-ß-coniferyl aldehyde ether (13), trans-p-coumaryl aldehyde (14), coniferyl aldehyde (15), 5,7-dihydroxychromone (16), trans-3,5-dimethoxy-4-hydroxy-cinnamic aldehyde (17), frambinone (18), and alternariol 4-methyl ether (19). Compounds 8-10,14,18,19 were firstly isolated from Verbenaceae family, compound 13 was obtained from Vitex species, and 6,7,12,15-17 from V. negundo var. cannabifolia for the first time, respectively. The isolated compounds were evaluated for their anti-inflammatory and cytotoxic effects in vitro. Eight compounds (3,5,7,10,11,14,15,17) showed inhibition against NO production in LPS-stimulated RAW 267.4 cells (IC50 in the range of 7.8-81.1 µmol•L⁻¹) and four compounds (1-4) showed cytotoxicity on HepG-2 cells (IC50 in the range of 5.2-24.2 µmol•L⁻¹).

[Absorption characteristics of 5 components of Xinshao extract in Caco-2 cells].

To study the absorption characteristics of Xinshao extracts in Caco-2 cells. In this paper, human colon adenocarcinoma cell line Caco-2 cell model was established, and UPLC-MS method was applied to determinate the contents of five components of Xinshao extracts(albiflorin, gallic acid, caffeic acid, scutellarin and apigenin-7-O-glucronide) in cell lysates. This model was also used to study the effect of different drug concentrations, pH, time and temperature on the absorption of five components, investigate the transport of the five components of Xinshao extracts under the conditions with or without P-glycoprotein inhibitors, and predict the absorption mechanism of these five components in Caco-2 cells. The experimental results showed that the absorption of five components of Xinshao extracts in Caco-2 cells was time-dependent at 37 ℃, and concentration-dependent in the range of 0.5-12.5 g•L⁻¹, with a passive diffusion mechanism. At the pH of 4-7.4, the absorption of caffeic acid, scutellarin and apigenin-7-O-glucronide was significantly declined with the increase of pH(P<0.05). At the temperature of 4 to 37 ℃, the absorption of caffeic acid was declined with the increase of temperature, while the absorption of other four components was increased with the increase of temperature. Compared with the control group, caffeic acid and scutellarin cell absorption was significantly higher(P<0.05) after treatment with P-glycoprotein inhibitors(verapamil and cyclosporine A). The results indicated that, the absorption mechanism of five components in Xinshao extracts may be of passive diffusion, and the caffeic acid and scutellarin may be the substrates of P-glycoprotein.

Anti-inflammatory Labdane Diterpenoids from Leonurus macranthus.

Twenty new polyoxygenated labdane diterpenoids (1-20) were isolated from the aerial parts of Leonurus macranthus. Their structures were elucidated on the basis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The absolute configurations of macranthin A (1) and 6-O-deacetylmacranthin A (2) were determined by single-crystal X-ray crystallographic analysis and a modified Mosher's method, respectively. Compounds 1-9, 12, 14, and 19 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells with IC50 values of 10.0-63.7 µM.

[Oil of Piper longum unsaponifiable matter prevents cholesterol gallstone formation].

To observe the effect of various doses of oil of Piper longum unsaponifiable matter (OPUM) to cholesterol gallstones in experimental mice. C57BL/6 mice (n = 60) were randomly divided into 6 groups: control group, model group, OPUM (15, 30 and 60 mg x kg(-1)) group and ursodeoxycholic acid (UDCA, 60 mg x kg(-1)) group, administered for 10 weeks. The level of serum lipid and liver function enzymes were tested. The gallbladder was removed and bile was obtained by centrifugation. Next, the levels of the bile total cholesterol (TC), phospholipid (PL) and bile acid (TBA) were measured. The indicators of lipid peroxidation were determined and cholesterol saturation index (CSI) was calculated. The liver histological changes were observed by HE staining. The results showed that serum TC, TG (triglycerides) and AST (aspartate transaminase) contents, gallbladder cholesterol crystallization and CSI increased significantly (P < 0.05). In addition, the activity of SOD decreased significantly and MDA content increased significantly in liver (P < 0.05). HE staining results showed that the hepatic cord disorder and intracellular lipid droplets increased significantly. All results indicate that lithogenic diet lead to the formation of cholesterol gallstones. In OPUM (30 and 60 mg x kg(-1)) group, serum TC, TG and AST content, gallbladder cholesterol crystallization and CSI decreased significantly, the activity of SOD increased significantly and MDA content decreased significantly. HE staining results showed that OPUM can improve the morphology of liver cell, reduce the degree of hepatic cord disorders and restore the cell morphology close to normal. The cause of OPUM prevents cholesterol gallstone formation maybe due to protect the integrity of the liver cells, lower CSI, and reduce cholesterol crystal formation and hence prevent cholesterol gallstone formation.

[Clinical analysis of 8 cases with cryoglobulinemic glomerulonephritis].

OBJECTIVE: To study the clinical and pathological features of cryoglobulinemic glomerulonephritis. METHODS: The clinical and pathologic data from 8 cases with cryoglobulinemic glomerulonephritis, which were referred to Peking University First Hospital from 2002 to 2006, were reviewed. RESULTS: There were seven males and one female, with an average age of 48.6 +/- 13.6 years. Most of the patients were referred as primary glomerulonephritis and the mean interval from onset to diagnosis was 19 months. 5 cases showed positive result with blood cryoglobulin test as inferred from renal biopsy. At the time of diagnosis 5 cases (5/8) showed nephritic syndrome and 3 (3/8) progression of renal failure. Fifty percent of the patients died in 6 months during the two-year follow-up. The main pathological pattern was membranoproliferative glomerulonephritis (7/8) with glomerular subendothelial deposits and intraluminal thrombi. Four cases (4/8) were associated with lymphoproliferative disorders and only one case with HCV infection. CONCLUSION: Cryoglobulinemic glomerulonephritis was not rare. Most of the patients were diagnosed at a late stage with a poor prognosis in China. It should draw a special attention of the clinicians.