Effects of Tylophora yunnanensis Schltr on regulating the gut microbiota and its metabolites in non-alcoholic steatohepatitis rats by inhibiting the activation of NOD-like receptor protein 3.

ETHNOPHARMACOLOGICAL RELEVANCE: Tylophora yunnanensis Schltr (TYS) is widely distributed in Yunnan, Guizhou, and other places in China. It is commonly used by folks to treat hepatitis and other liver-related diseases; however, its mechanism of action is still unclear. AIM OF THE STUDY: This study aimed to determine the effects of TYS on regulating gut microbiota and its metabolites in non-alcoholic steatohepatitis (NASH) rats by inhibiting the activation of NOD-like receptor protein3 (NLRP3). MATERIAL AND METHODS: An HFD-induced rat model was established to investigate if the intragastric administration of TYS could mediate gut microbiota and their metabolites to ultimately improve the symptoms of NASH. The improving effects of TYS on NASH rats were assessed by measuring their body weight, lipid levels, histopathology, and inflammatory factor levels in the rat models. The regulatory effects of TYS on NLRP3 in the NASH rats were analyzed using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), which determined the levels of NLRP3-related factors. The changes in the composition of the gut microbiota of NASH rats were analyzed using 16S rRNA gene sequencing technology. Meanwhile, the Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for the non-targeted analysis of metabolites in the cecum contents. RESULTS: The results showed that TYS could improve NASH by decreasing the body weight and levels of lipid, AST, ALT, LPS, FFA, VLDL, IL-1ß, IL-6, TNF-α, TGF-ß, NLRP3, ASC, and Caspase-1 in the NASH rats. The analysis of gut microbiota showed that TYS could improve the diversity and abundance of gut microbiota and alter their composition by decreasing the Firmicutes/Bacteroidetes (F/B) ratio and relative abundances of Lachnospiraceae, Christensenellaceae, Blautia, etc. while increasing those of Muribaculaceae, Rumiaococcus, Ruminococcaceae, etc. The analysis of metabolites in the cecum contents suggested that the arachidonic acid metabolism, bile secretion, serotonergic synapse, Fc epsilon RI signaling pathway, etc. were regulated by TYS. The metabolites enriched in these pathways mainly included chenodeoxycholic acid, prostaglandin D2, TXB2, 9-OxoODE, and 13(S)-HOTrE. CONCLUSIONS: These findings suggested that TYS could alleviate the NASH symptoms by decreasing the body weight, regulating the lipid levels, reducing the inflammatory response, and inhibiting the expression levels of NLRP3, ASC, and Caspase-1 in the NASH rats. The changes in the composition of gut microbiota and their metabolic disorder were closely related to the activation of NLRP3. TYS could significantly inhibit the activation of NLRP3 and regulate the composition of gut microbiota and the disorder of metabolites during NASH modeling.

The potential benefits and mechanisms of protein nutritional intervention on bone health improvement.

Osteoporosis commonly occurs in the older people and severe patients, with the main reason of the imbalance of bone metabolism (the rate of bone resorption exceeding the rate of bone formation), resulting in a decrease in bone mineral density and destruction of bone microstructure and further leading to the increased risk of fragility fracture. Recent studies indicate that protein nutritional support is beneficial for attenuating osteoporosis and improving bone health. This review summarized the classical mechanisms of protein intervention for alleviating osteoporosis on both suppressing bone resorption and regulating bone formation related pathways (promoting osteoblasts generation and proliferation, enhancing calcium absorption, and increasing collagen and mineral deposition), as well as the potential novel mechanisms via activating autophagy of osteoblasts, altering bone related miRNA profiles, regulating muscle-bone axis, and modulating gut microbiota abundance. Protein nutritional intervention is expected to provide novel approaches for the prevention and adjuvant therapy of osteoporosis.

Walnut green husk ethanol extract improves gut microbiota and their metabolites associated with NLRP3 in non-alcoholic steatohepatitis.

Increasing studies have shown that walnut green husk (WGH) has obvious effects on reducing lipid, resisting oxidation, and protecting the liver. However, the mechanism by which WGH can prevent high-fat diet (HFD)-induced non-alcoholic steatohepatitis (NASH) remains unclear. This study is aimed at investigating the effects of WGH ethanol extract (WGHE) on NLRP3-related biochemical indicators and the diversity and metabolism of gut microbiota in HFD-induced NASH rats. WGHE was administered to HFD-induced NASH rats for 6 weeks. The results showed that WGHE could decrease the levels of blood and liver TC, TG, LDL-C, AST, and ALT and the levels of liver indices, including IL-1ß, IL-6, TNF-α, TGF-ß, FFA, VLDL, caspase-1, ASC, and NLRP3, while it could increase the levels of HDL-C. The pathological damage to liver tissues was significantly reduced. Moreover, WGHE could reduce the Firmicutes/Bacteroidetes (F/B) ratio and the relative abundances of potentially harmful bacteria, such as Lachnospiraceae and Christensenellaceae, and increase that of potentially beneficial bacteria, such as norank_f__Muribaculaceae. These bacteria were associated with NASH and most of them were significantly associated. A total of 23 gut bacteria and 31 metabolites were significantly altered by HFD, which was reversed by WGHE. The common functional pathways, including lipid metabolism and steroid biosynthesis, were identified through the analysis of KEGG metabolic pathways. In addition, the changes in gut microbiota, such as unclassified_f__Lachnospiraceae, unclassified_g__Blautia, and unclassified_g__Desulfovibrio, were associated with the changes in key intestinal metabolites, such as arachidonoyl amine, xanthine, and 25,26-epoxy-1α-hydroxyvitamin D3. In conclusion, WGHE could mitigate HFD-induced NASH in rats by interfering with the NLRP3-related gut microbiota and their metabolites.

Application of immune checkpoint targets in the anti-tumor novel drugs and traditional Chinese medicine development.

Immune checkpoints are the crucial regulators of immune system and play essential roles in maintaining self-tolerance, preventing autoimmune responses, and minimizing tissue damage by regulating the duration and intensity of the immune response. Furthermore, immune checkpoints are usually overexpressed in cancer cells or noninvasive cells in tumor tissues and are capable of suppressing the antitumor response. Based on substantial physiological analyses as well as preclinical and clinical studies, checkpoint molecules have been evaluated as potential therapeutic targets for the treatment of multiple types of cancers. In the last few years, extensive evidence has supported the immunoregulatory effects of traditional Chinese medicines (TCMs). The main advantage of TCMs and natural medicine is that they usually contain multiple active components, which can act on multiple targets at the same time, resulting in additive or synergistic effects. The strong immune regulation function of traditional Chinese medicine on immune checkpoints has also been of great interest. For example, Astragalus membranaceus polysaccharides can induce anti-PD-1 antibody responses in animals, and these antibodies can overcome the exhaustion of immune cells under tumor immune evasion. Furthermore, many other TCM molecules could also be novel and effective drug candidates for the treatment of cancers. Therefore, it is essential to assess the application of immune checkpoints in the development of new drugs and TCMs. In this review, we focus on research progress in the field of immune checkpoints based on three topics: (1) immune checkpoint targets and pathways, (2) development of novel immune checkpoint-based drugs, and (3) application of immune checkpoints in the development of TCMs.

Circulating fatty acids and risk of gestational diabetes mellitus: prospective analyses in China.

OBJECTIVE: We aimed to examine prospective associations between circulating fatty acids in early pregnancy and incident gestational diabetes mellitus (GDM) among Chinese pregnant women. METHODS: Analyses were based on two prospective nested case-control studies conducted in western China (336 GDM cases and 672 matched controls) and central China (305 cases and 305 matched controls). Fasting plasma fatty acids in early pregnancy (gestational age at enrollment: 10.4 weeks(s.d., 2.0)) and 13.2 weeks (1.0), respectively) were determined by gas chromatography-mass spectrometry, and GDM was diagnosed based on the International Association of Diabetes in Pregnancy Study Groups criteria during 24-28 weeks of gestation. Multiple metabolic biomarkers (HOMA-IR (homeostatic model assessment for insulin resistance), HbA1c, c-peptide, high-sensitivity C-reactive protein, adiponectin, leptin, and blood lipids) were additionally measured among 672 non-GDM controls at enrollment. RESULTS: Higher levels of saturated fatty acids (SFAs) 14:0 (pooled odds ratio, 1.41 for each 1-s.d. increase; 95% CI: 1.25, 1.59) and 16:0 (1.19; 1.05, 1.35) were associated with higher odds of GDM. Higher levels of n-6 polyunsaturated fatty acid (PUFA) 18:2n-6 were strongly associated with lower odds of GDM (0.69; 0.60, 0.80). In non-GDM pregnant women, higher SFAs 14:0 and 16:0 but lower n-6 PUFA 18:2n-6 were generally correlated with unfavorable metabolic profiles. CONCLUSIONS: We documented adverse associations of 14:0 and 16:0 but a protective association of 18:2n-6 with GDM among Chinese pregnant women. Our findings highlight the distinct roles of specific fatty acids in the onset of GDM.

Microdesigned Nanocellulose-Based Flexible Antibacterial Aerogel Architectures Impregnated with Bioactive Cinnamomum cassia.

This work is strategically premeditated to study the potential of a herbal medicinal product as a natural bioactive ingredient to generate nanocellulose-based antibacterial architectures. In situ fibrillation of purified cellulose was done in cinnamon extract (ciE) to obtain microfibrillated cellulose (MFC). To this MFC suspension, carboxylated cellulose nanocrystals (cCNCs) were homogeneously mixed and the viscous gel thus obtained was freeze-dried to obtain lightweight and flexible composite aerogel architectures impregnated with ciE, namely, ciMFC/cCNCs. At an optimal concentration of 0.3 wt % cCNCs (i.e., for ciMFC/cCNCs_0.3), an improvement of around 106% in compressive strength and 175% increment in modulus were achieved as compared to pristine MFC architecture. The efficient loading and interaction of ciE components, specifically cinnamaldehyde, with MFC and cCNCs resulted in developing competent antibacterial surfaces with dense and uniform microstructures. Excellent and long-term antimicrobial activity of the optimized architectures (ciMFC/cCNCs_0.3) was confirmed through various antibacterial assays like the zone inhibition method, bacterial growth observation at OD600, minimum inhibitory concentration (MIC, here 1 mg/mL), minimum bactericidal concentration (MBC, here 3-5 mg/mL), and Live/Dead BacLight viability tests. The changes in the bacterial morphology with a disrupted membrane were further confirmed through various imaging techniques like confocal laser scanning microscopy, FESEM, AFM, and 3D digital microscopy. The dry composite architecture showed the persuasive capability of suppressing the growth of airborne bacteria, which in combination with antibacterial efficiency in the wet state is considered as an imperative aspect for a material to act as the novel biomaterial. Furthermore, these architectures demonstrated excellent antibacterial performance under real "in use" contamination prone conditions. Hence, this work provides avenues for the application of crude natural extracts in developing novel forms of advanced functional biomaterials that can be used for assorted biological/healthcare applications such as wound care and antimicrobial filtering units.

Bio-extract amalgamated sodium alginate-cellulose nanofibres based 3D-sponges with interpenetrating BioPU coating as potential wound care scaffolds.

In this work, sodium alginate (SA) based "all-natural" composite bio-sponges were designed for potential application as wound care scaffold. The composite bio-sponges were developed from the aqueous amalgamation of SA and cellulose nanofibres (CNFs) in bio-extracts like Rice water (Rw) and Giloy extract (Ge). These sponges were modified by employing a simple coating strategy using vegetable oil-based bio-polyurethane (BioPU) to tailor their physicochemical and biological properties so as to match the specific requirements of a wound care scaffold. Bio-sponges with shared interpenetrating polymeric network structures were attained at optimized BioPU coating formulation. The interpenetration of BioPU chains within the sponge construct resulted in the formation of numerous micro-networks in the interconnected microporous structure of sponges (porosity ≥75%). The coated sponge showed a superior mechanical strength (compressive strength ~3.8 MPa, compressive modulus ~35 MPa) with appreciable flexibility and recoverability under repeated compressive loading-unloading cycles. A tunable degradation behaviour was achieved by varying BioPU coating concentrations owing to the different degree of polymer chain entanglement within the sponge construct. The physical entanglement of BioPU chains with core structural components of sponge improved their structural stability by suppressing their full fragmentation in water-based medium without affecting its swelling behaviour (swelling ratio > 1000%). The coated sponge surface has provided a suitable moist-adherent physical environment to support the adhesion and growth of skin cells (HaCaT cells). The MTT (3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide) assay and hemolytic assay revealed the non-toxic and biocompatible nature of coated sponges in vitro. Moreover, no signs of skin erythema or edema were observed during in vivo dermal irritation and corrosion test performed on the skin of Sprague Dawley (SD) rats. Our initial observations revealed the credibility of these sponges as functional wound care scaffolds as well as its diverse potential as a suitable substrate for various tissue engineering applications.

CuS-NiS2 nanomaterials for MRI guided phototherapy of gastric carcinoma via triggering mitochondria-mediated apoptosis and MLKL/CAPG-mediated necroptosis.

Gastric carcinoma is one of the most lethal malignant tumors. As part of our long-term efforts on seeking effective diagnosis and therapeutic strategies of gastric cancer, we present herein novel ternary copper-based chalcogenide nanoplatform CuS-NiS2 nanomaterials with outstanding photothermal (PT)/photodynamic (PD) property that could effectively suppress human gastric cancer in vitro and in vivo without obvious side effects. We revealed that CuS-NiS2 induced reactive oxygen species (ROS) generation, leading to apoptosis through Bcl-2/Bax pathway of human gastric cancer cells under 808 nm near-infrared (NIR) irradiation. In addition, we also confirmed that the combination of CuS-NiS2 and 808 nm NIR laser treatment triggered necroptosis by regulating the novel pathway MLKL/CAPG of human gastric cancer cells. Moreover, the CuS-NiS2 exhibited excellent contrast enhancement according to magnetic resonance imaging (MRI). Taken together, we reported new ternary copper-based chalcogenide nanomaterials CuS-NiS2, which could be successfully applied for MRI-guided PT/PD therapy of gastric carcinoma through mitochondria-mediated apoptosis and MLKL/CAPG-mediated necroptosis.

Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage.

Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.

Comparative Transcriptome Analysis Reveals Differentially Expressed Genes and Signaling Pathways Between Male and Female Red-Tail Catfish (Mystus wyckioides).

Sexual dimorphism is widespread in fish species. The red-tail catfish (Mystus wyckioides) is a commercially important catfish in the lower reaches of the Lancang River and the Mekong basin, and it shows a growth advantage in males. Here, RNA-seq was for the first time used to explore the gene expression difference between the sexes in the hypothalamus and pituitary of red-tail catfish, respectively. In the hypothalamus, 5732 and 271 unigenes have significantly higher and lower expressions, respectively, in males compared with females. KEGG analysis showed that 212 DEGs were annotated to 216 signaling pathways, and enrichment analysis suggested different levels of cAMP and glutamatergic synapse signaling between male and female hypothalami and some of the DEGs appear involved in gonad development and growth. In the pituitary, we found only 19 differentially expressed unigenes, which were annotated to 32 signaling pathways, most of which play important roles in gonad development.

Kaempferol's Protective Effect on Ethanol-Induced Mouse Primary Hepatocytes Injury Involved in the Synchronous Inhibition of SP1, Hsp70 and CYP2E1.

The mechanism of ethanol-induced hepatotoxicity was complicated, accompanied by the over-expressions of the cytochrome P450 2E1 (CYP2E1), heat shock protein 70 (Hsp70) and the nuclear factor specificity protein 1 (SP1). Kaempferol (Kaem) could protect the ethanol-induced hepatotoxicity likely by inhibiting the CYP2E1 expression and activity. This study investigated the protective mechanism(s) of kaempferol on ethanol-induced toxicity by dynamic alteration of SP1, Hsp70 and CYP2E1 among the nucleus and different organelles in hepatocytes. After ethanol treatment alone and co-incubation hepatocytes with kaempferol, protein levels of CYP2E1, Hsp70, and SP1 were determined in vitro (western blotting and immunofluorescence). Hepatocytes' viability was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods. Glutathione (GSH) levels were evaluated for ethanol-induced oxidative stress. In the ethanol-treated hepatocytes, kaempferol decreased protein levels of CYP2E1 in both microsome and mitochondria, cytosolic Hsp70 and SP1 in nuclear and cytosol, and the oxidative stress and increased the cell viability compared to those of ethanol group. Collectively, our findings propose that the protective mechanism of kaempferol is involved in the synchronous, early and persistent inhibitions of mitochondrial and microsomal CYP2E1, cytosolic Hsp70 and nuclear and cytosolic SP1 in mouse primary hepatocytes' injury induced by ethanol.

Echinacoside Increases Sperm Quantity in Rats by Targeting the Hypothalamic Androgen Receptor.

Male infertility is a major health issue with an estimated prevalence of 4.2% of male infertility worldwide. Our early work demonstrated that Cistanche extracts protect against sperm damage in mice and that echinacoside (ECH) is one of the major active components. Here we report an essential role for ECH, a natural product that reverses or protects against oligoasthenospermia in rats. ECH was assayed by HPLC, the quantity and quality of sperm was evaluated and hormone levels were determined by radioimmunosorbent assay. ECH reduced levels of androgen receptor (AR) and key steroidogenic-related genes as determined by Western blot and qPCR analysis. The interaction between ECH and AR were evaluated by indirect ELISA and molecular docking. The results show that ECH combined with hypothalamic AR in the pocket of Met-894 and Val-713 to inhibit transfer of AR from the cytoplasm to nuclei in the hypothalamus. While negative feedback of sex hormone regulation was inhibited, positive feedback was stimulated to increase the secretion of luteinizing hormone and testosterone subsequently enhancing the quantity of sperm. Taken together, these data demonstrate that ECH blocks AR activity in the hypothalamus to increase the quantity of sperm and protect against oligoasthenospermia in rats.

[Research progress of puerarin and its derivatives on anti-inflammatory and anti-gout activities].

The research progress of puerarin and its derivatives in anti-inflammatory and anti-gout activities was reviewed in this paper. Puerarin possesses anti-inflammatory activity by affecting immunocyte, inflammation cytokines and signaling pathway. Puerarin also has anti-gout activity through inhibition of xanthine oxidase, promoting the excretion of uric acid to reduce serum uric acid level. Although its ability in reducing uric acid level was lower than that of allopurinol in clinical application, puerarin can also enhance the total antioxidant and free radical scavenging with stronger anti-inflammatory effect, so it will be a promising research direction to find new drugs with better anti-gout activity and less side effects by modifying the chemical structure of puerarin.

Echinacoside and Cistanche tubulosa (Schenk) R. wight ameliorate bisphenol A-induced testicular and sperm damage in rats through gonad axis regulated steroidogenic enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: Male infertility has been increasing over the last decades and being a pressing health problem nowadays. Cistanche tubulosa (CT) is a traditional Chinese medicine used to boost male sexual function. Echinacoside (ECH) is one of the major compounds exist in CT and might be a potential agent to protect testis and sperm injury. AIM OF THE STUDY: To investigate the mechanisms behind the possible protective effects of CT and ECH against testicular and sperm toxicity. MATERIALS AND METHODS: CT was identified by 5.8s gene sequencing. The major compositions (echinacoside and acteoside) of CT were quantified by HPLC method. The adult male Sprague-Dawley rats were exposed to BPA, CT or ECH for 42 consecutive days. The sperm parameters were observed by dark-field microscope; serum hormone levels (FSH, LH and testosterone) were tested by radio immunosorbent; LDH-x activity were evaluated using commercial kits; the expressions of the key steroidogenic enzymes were evaluated by qRT-PCR, heat map, immunofluorescence and western blot. RESULTS: The CT and ECH treatments against BPA-induced testicular and sperm toxicity showed that CT and ECH have reversed BPA-induced abnormality in sperm characteristics, testicular structure and normalized serum testosterone. This was concomitant with the increased expression of LDH-x as well as the key steroidogenic enzymes including StAR, CYP11A1, 3ß-HSD, 17ß-HSD and CYP17A1, suggesting that CT and ECH enhanced testosterone biosynthesis. CONCLUSIONS: CT and ECH attenuated poor sperm quality and testicular toxicity in rats through up-regulation steroidogenesis enzymes and ECH is the active compound of CT as a potential natural reproductive agent.

Study on the Antinociceptive Effects of Herba Epimedium in Mice.

The present study was conducted to investigate the antinociceptive action of relationship between Herba Epimedium (HE) and 5-HT1A receptor, between Herba Epimedium (HE) and 5-HT2A receptor. We used the hot-plate method and the writhing assay in mice by the intracerebroventricular (i.c.v.) injection and observed the analgesic effect of HE. Furthermore, through the i.c.v. injection, 5-HT1A receptor partial agonist Buspirone, antagonist Propranolol, the adrenaline ß 1-receptor selective blocking agent Metoprolol, and 5-HT2A receptor agonist hydrochloride DOI and antagonist Ketanserin were used, and, 5 min later, HE was used to investigate the impacts of drugs on the analgesic effect in the same way. Results showed that HE had fast and significant antinociception in nervous system, and the effects can persist for a long time. Buspirone and Hydrochloride DOI can remarkably increase the antinociception of HE in nervous system. Ketanserin leads to a significant decrease in its antinociception in nervous system; Metoprolol also has antinociceptive action in nervous system, but it can inhibit the antinociceptive effect of Herba Epimediumin peripheral region. These results suggest that HE has significant antinociception effect and its mechanism is related with 5-HT1A receptor, 5-HT2A receptor, and adrenaline ß 1-receptor.

[Study on hemolytic mechanism of polyphyllin II].

To study the hemolytic effect of polyphyllin II (PP II) mediated by anion channel protein and glucose transporter 1 (GLUT1), in order to initially reveal its hemolytic mechanism in vitro. In the experiment, the spectrophotometric method was adopted to detect the hemolysis of PP II in vitro and the effect of anion channel-related solution and blocker, glucose channel-related inhibitor and multi-target drugs dehydroepiandrosterone (DHEA) and diazepam on the hemolysis of PP II. The scanning electron microscope and transmission electron microscope were used to observe the effect of PP II on erythrocyte (RBC) morphology. The results showed that PP II -processed blood cells were severely deformed into spherocytes, acanthocyturia and vesicae. According to the results of the PP II hemolysis experiment in vitro, the anion hypertonic solution LiCl, NaHCO3, Na2SO4 and PBS significantly inhibited the hemolysis induced by PP II (P < 0.05), while blockers NPPB and DIDS remarkably promoted it (P < 0.01). Hyperosmotic sodium chloride, fructose and glucose at specific concentrations notably antagonized the hemolysis induced by PP II (P < 0.05). The glucose channel inhibitor Cytochalasin B and verapamil remarkably antagonized the hemolysis induced by PP II (P < 0.01). The hemolysis induced by PP II could also be antagonized by 1 gmol x L(1) diazepam and 100 µmol x L(-1) DHEA pretreated for 1 min (P < 0.01). In conclusion, the hemolytic mechanism of PP II in vitro may be related to the increase in intracellular osmotic pressure and rupture of erythrocytes by changing the anion channel transport activity, with GLUT1 as the major competitive interaction site.

[Influence of different penetration enhancers on Lappaconitine transcutaneous permeation].

OBJECTIVE: To investigate the effects of different kinds and concentration of transdermal enhancers on Lappaconitine transcutaneous permeation when used individually or together. METHOD: Using modified Franz-type diffusion cell and excised human body skin as an in vitro transdermal model, the concentration of lappaconitine was determined by HPLC, then cumulative permeation quantity (Q) and stability rate (J) of progesterone were calculated. RESULT: Penetration enhancers such as propylene glycol, dodecanol, IPM, and particularly 3% OA and Azone, can significantly enhance the penetration rate of lappaconitine. Concentration effect of penetration enhancers concentration on lappaconitine transcutaneous permeation were found in experiments, the permeation effect of Azone was better than Azone + OA and Azone + propylene glycol. CONCLUSION: The transdermal rate of lappaconitine from batch which contains 3% OA or Azone is higher than others. Combination of Azone with other penetration enhancers is not recommended for Lappaconitine transcutaneous permeation.

Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay.

We developed a functional cell-based high-throughput screening (HTS) assay to identify modulators of the human neuromedin subtype 2 receptor. This assay utilized the signal transduction pathway of hNMU2R, which is positively coupled to adenylyl cyclase and downstream calcium signal pathways. We describe in detail a robust, sensitive, and functional assay for the hNMU2R G-protein-coupled receptor expressed in human embryonic kidney (HEK)-293 cells, whose activity was reflected by a luciferase reporter gene transcriptionally regulated by a 3-repeat serum response element (SRE)-3 repeat multiple response element (MRE)-3 repeat cyclic AMP (cAMP) response element (CRE)-VIP mini promoter. The HEK 293 clonal cell line, stably co-transfected with the 3xSRE/3xMRE/3xCRE/VIP mini promoter-driven luciferase and pCDNA3.1-NMU2R plasmid, was selected by active geneticin sulfate and their ability to express luciferase with a forskolin challenge following hNMU plus forskolin, known to activate intracellular signal transduction. Then the cell density, incubation time, dimethyl sulfoxide (DMSO) concentration used to screen the hNMU receptor subtype 2 specific agonist were optimized, and whether intrinsic luminescent substance of extracts isolated from traditional Chinese herbs disturbs luminescence of luciferase expressed in HEK293 cells was considered. The optimal incubation time was found to be between 8 and 9h, the cell density and DMSO concentrations were optimized from 3x10(4) to 6x10(4), and less than 2%, respectively. Our data show that hNMU2R luci-HEK293 cells and their assay exhibit a low background and ideal model for high-throughput screening. These results demonstrate that this reporter gene assay is useful for pharmacological analysis, and is amenable to HTS for human NMU2R agonists.

[Lymph node micrometastases and expression of metastasis-related gene proteins in patients with colorectal cancer].

OBJECTIVE: To study lymph node micrometastases (LNMM), expression of nm23-H(1), MMP(9), TIMP(2) proteins, and their relationship and clinical significance in patients with stage Dukes B colorectal cancer. METHODS: Thirty patients with stage Dukes B colorectal cancer were studied. LNMM in these patients was detected by immunohistochemical anti-cytokeratin 20 (CK20) staining. The expression of nm23-H(1), MMP(9) and TIMP(2) proteins in primary tumors was examined by Strept-avidin-biotin complex method. Clinical-pathological data and survival of each patient were recorded and analyzed. RESULTS: (1) The positive dyeing of CK20 was observed in 26.7% for cases and in 7.8% for lymph nodes of 30 patients with stage Dukes B colorectal cancer. (2) Different expression of nm23-H(1) and MMP(9) proteins in the patients between stage Dukes B and stage Dukes CD was observed (P < 0.05). The decreased nm23-H(1) expression, and/or the increased MMP(9) expression in primary stage Dukes B tumors were significantly associated with LNMM (P < 0.05). Sensitivity and specificity for detection of LNMM by using nm23-H(1) or MMP(9) were respectively 62.5% and 81.8% or 75.0% and 69.8%. If by combining nm23-H(1) with MMP(9), specificity for detection of LNMM became 90.9%. The expression of TIMP(2) protein was not related with stage Dukes and LNMM. (3) The percent of tumor recurrence and/or metastasis for the stage Dukes B patients with LNMM was significantly higher than that for the patients without LNMM (P < 0.05), but the survival percent for the patients with LNMM was significantly lower than that for the patients without LNMM. The outcome for the patients with nm23-H(1) (-) LNMM (+) or MMP(9) (+) LNMM (+) was significantly worse than that for patients with nm23-H(1) (+) LNMM (-) or MMP(9) (+) LNMM (-) (P < 0.05). CONCLUSIONS: LNMM is detected by immunohistochemical anti-CK20 staining. The expression of nm23-H(1) and MMP(9) in primary stage Dukes B tumors was significantly associated with LNMM. The outcome in the LNMM patients with nm23-H(1) (-) and/or MMP(9) (+) were worse. Combining examination of CK20 for lymph nodes with expression of nm23-H(1) and MMP(9) for primary tumors is of important clinical significance for staging of Dukes, selection of adjuvant treatment and evaluation of prognosis in patients with colorectal cancer.