Amelioration of TPA-induced skin inflammation by the leaf extract of Vernonia amygdalina involves ERK/STAT3 (Ser727) signaling inhibition.

BACKGROUND: Uncontrolled inflammation causes health problems. Extracellular signal-regulated kinase (ERK) phosphorylates signal transducer and activator of transcription 3 (STAT3) at Ser727, resulting in inflammation. The leaf of Vernonia amygdalina (VA) is a medicinal herb for managing inflammation-associated diseases. Oral administration or topical application of VA leaf extract exerts anti-inflammatory effects in rat models. However, the anti-inflammatory mechanisms of the herb are not fully understood. PURPOSE: In this study, we aimed to investigate the involvement of ERK/STAT3 (Ser727) signaling in the anti-inflammatory effects of an ethanolic extract of VA leaves. STUDY DESIGN AND METHODS: Extracts of VA leaves were prepared with different concentrations of ethanol. A LPS-stimulated RAW264.7 cell model was used for in vitro assays, and a TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model was employed for in vivo assays. The 95% ethanol extract of VA leaves (VAE) exerted the strongest inhibitory effect on nitric oxide (NO) production in LPS-stimulated macrophages; thus it was selected for use in this study. Hematoxylin and eosin (H&E) staining was used to examine pathological conditions of mouse ear tissues. Griess reagent was employed to examine NO generation in cell cultures. Immunoblotting and ELISA were used to examine protein levels, and RT-qPCR was employed to examine mRNA levels. RESULTS: Topical application of VAE ameliorated mouse ear edema induced by TPA. VAE suppressed the phosphorylation of ERK (Thr202/Tyr204) and STAT3 (Ser727); and decreased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, IL-1ß and tumor necrosis factor-α (TNF-α) in the mouse ear tissues and in LPS-stimulated RAW 264.7 cells. VAE also inhibited NO production, and lowered mRNA levels of IL-6, IL-1ß and TNF-α in the macrophages. CONCLUSIONS: VAE ameliorates TPA-induced mouse ear edema. Suppression of ERK/STAT3 (Ser727) signaling is involved in VAE's anti-inflammatory effects. These novel data provide further pharmacological justifications for the medicinal use of VA in treating inflammation-associated diseases, and lay the groundwork for developing VAE into a new anti-inflammatory agent.

Biological Response Profiling Reveals the Functional Differences of Main Alkaloids in Rhizoma Coptidis.

Rhizoma Coptidis (RC) is a widely used traditional Chinese medicine. Although modern research has found that some alkaloids from RC are the pharmacologically active constituents, the differences in their biological effects are not completely clear. This study analyzed the differences in the typical alkaloids in RC at a systematic level and provided comprehensive information on the pharmaceutical mechanisms of the different alkaloids. The ethanol RC extract (RCE) was characterized using HPLC assay. HepG2, 3T3-L1, and RAW264.7 cells were used to detect the cytotoxicity of alkaloids. Transcriptome analyses were performed to elucidate the cellular pathways affected by RCE and alkaloids. HPLC analysis revealed that the typical alkaloids of RCE were berberine, coptisine, and palmatine. Coptisine and berberine displayed a stronger inhibitory effect on cell proliferation than palmatine. The overlapping ratios of differentially expressed genes between RCE and berberine, coptisine, and palmatine were 70.8%, 52.6%, and 42.1%, respectively. Pathway clustering analysis indicated that berberine and coptisine possessed a certain similarity to RCE, and both compounds affected the cell cycle pathway; moreover, some pathways were uniquely enriched by berberine or coptisine. Berberine and coptisine had different regulatory effects on genes involved in lipid metabolism. These results provide comprehensive information on the pharmaceutical mechanisms of the different RC alkaloids and insights into their better combinatory use for the treatment of diseases.

Effects of a homogeneous polysaccharide from Sijunzi decoction on human intestinal microbes and short chain fatty acids in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Sijunzi decoction (SJZD) is a classic recipe in traditional Chinese medicine (TCM) to strengthen the spleen and replenish Qi. It is well known for treating disorders of gastrointestinal function manifested in poor appetite, reduced food intake and loose stools. Polysaccharide is the most abundant constituent and the major effective component in SJZD. AIM OF THE STUDY: The present study aimed to understand the immunomodulatory mechanism of S-3-1, a homogeneous polysaccharide purified from SJZD with immune-enhancement activity, by investigating its effects on human intestinal microbes and short chain fatty acids. MATERIALS AND METHODS: S-3-1 was incubated with simulated gastric juice, intestinal juice, and human fecal microflora independently and sequentially. The concentrations of total polysaccharide and reducing sugar were measured to identify the stability of independently and sequentially incubated S-3-1 in three in vitro fermentation models. Gas chromatograph (GC) analysis was used to measure the short chain fatty acid (SCFA) contents in human fecal samples. The human gut microbiota composition was measured by 16S rRNA gene Illumina MiSeq sequencing (V3-V4 region). RESULTS: S-3-1 was degraded in three in vitro fermentation models separately and sequentially. Both S-3-1 and incubated S-3-1 could regulate the abundances of Lactobacillus, Pediococcus, Streptococcus, Bacteroides, Enterococcus, Clostridium and Dorea in human intestinal microflora samples. Specifically, S-3-1 could only regulate the abundances of Paraprevotella and Oscillospira, while the influenced flora changed to Butyricimonas, Coprococcus, Dialister, Sutterella, Ruminococcus and Parabacteroides after sequential incubation of S-3-1. In contrast to S-3-1 showing no influence on the content of SCFA, incubated S-3-1 showed increased contents of acetic acid and total acid that were associated with its effects on the abundances of Enterococcus, Sutterella, Butyricimonas and Streptococcus. CONCLUSION: S-3-1 plays an immunomodulatory role by regulating the abundances of 9 intestinal bacteria genera. Incubated S-3-1 can regulate more bacteria genera, a total of 13 kinds, and can adjust the SCFA content to affect immunomodulation. Incubation with gastric and intestinal juices enhanced S-3-1's capability of modulating the intestinal flora composition and decreased the bacteria's need for a carbon source. This study could provide new insights for studies on the pharmacological mechanisms of polysaccharides in vitro.

Biological-Profiling-Based Systematic Analysis of Rhizoma Coptidis from Different Growing Regions and Its Anticholesterol Biosynthesis Activity on HepG2 Cells.

Rhizoma Coptidis is a widely cultivated traditional Chinese herb. Although the chemical profiles of Rhizoma Coptidis have been established previously, the biological profiling of Rhizoma Coptidis has not been conducted yet. In this study, we collected Rhizoma Coptidis varieties from four distinct growing regions and performed genome-wide biological response fingerprinting (BioReF) on HepG2 cells using a gene expression array. Similar biological pathways were affected by extracts of all four Rhizoma Coptidis varieties but not by their analogue, Mahoniae Caulis. Among these pathways, the terpenoid backbone biosynthesis pathway was highly enriched, and six genes in the mevalonate (MVA) pathway were all down-regulated. However, the expression, maturation, as well as the specific DNA binding capacity of their coordinate transcription factor, sterol response element binding protein 2 (SREBP2), was not affected by Rhizoma Coptidis extract (RCE) or its typical active alkaloid berberine. Cellular cholesterol content tests further verified the cholesterol-lowering function of RCE in vitro, which supplements evidence for the use of Rhizoma Coptidis in hyperlipidemia treatment. This is the first described example of evaluating the quality of Rhizoma Coptidis with BioReF and a good demonstration of using BioReF to uncover the mechanisms of herbs at a systematic level.

Purification, Preliminary Characterization, and Immunological Activity of Polysaccharides from Crude Drugs of Sijunzi Formula.

Sijunzi Decoction (SJZD) is a conventional prescription for curing spleen deficiency in Traditional Chinese Medicine and polysaccharide is its main ingredient. In order to explore the effective ingredients contributing to the immunological activity of SJZD, we isolated and purified seven homogeneous polysaccharides from Radix Ginseng (RS-3-1 and RS-3-2), Rhizoma Atractylodis Macrocephalae (BZ-3-1, BZ-3-2, and BZ-3-3), Poria (FL-3-1), and Radix Glycyrrhizae (GC-3-1) decoctions, respectively. The molecular weight of seven homogeneous polysaccharides ranged from 5.42 × 104 to 5.65 × 104 Da. Monosaccharide composition determined by GC-MS analysis showed that these polysaccharides were primarily composed of Rha, Ara, Xyl, Man, Glc, and Gal with various ratios. Immunological activity assay revealed that polysaccharides from four crude drug components of SJZD displayed inhibitory effects on the complement system. RS-3-1, BZ-3-1, FL-3-1, and GC-3-1 could significantly enhance the phagocytosis and increase the NO production and tumor necrosis factor (TNF-α) level in RAW 264.7 cells (p < 0.05). These results demonstrated the immunological activities of these polysaccharides from the four crude drugs. This study supports the therapeutic effect of SJZD in clinical use and is essential for further identification the immunopolysaccharide from SJZD decoction.

Curcumin pretreatment attenuates inflammation and mitochondrial dysfunction in experimental stroke: The possible role of Sirt1 signaling.

The effects of curcumin (CCM) on cerebral ischemia/reperfusion injury are not well understood. The aim of this study was to investigate whether CCM attenuates inflammation and mitochondrial dysfunction in a rat model of cerebral ischemia/reperfusion injury and whether Sirt1 is involved in these potential protective effects. Sirtinol, a Sirt1 inhibitor, was used to elucidate the underlying mechanism. Rats were subjected to 2h of transient middle cerebral artery occlusion (MCAO), followed by reperfusion for 24h. Brain magnetic resonance imaging (MRI) was used to detect infarct volumes. Neurological scores and brain water content were also assessed. Levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in the brain were detected using commercial enzyme-linked immunosorbent assay (ELISA) kits. Expression of SIRT1, acetylated p53 (Ac-p53), Bcl-2, and Bax was measured by western blotting. Our results suggested that CCM exerted a neuroprotective effect, as shown by reduced infarct volumes and brain edema and improved neurological scores. CCM also exerted anti-inflammatory effects, as indicated by decreased TNF-α and IL-6 levels in the brain. CCM elevated mitochondrial membrane potential, mitochondrial complex I activity, and mitochondrial cytochrome c levels, but reduced cytosolic cytochrome c levels. Moreover, CCM upregulated SIRT1 and Bcl-2 expression and downregulated Ac-p53 and Bax expression. These effects of CCM were abolished by sirtinol. In conclusion, our results demonstrate that CCM treatment attenuates ischemic stroke-induced brain injury via activation of SIRT1.

The Metabolism of Polysaccharide from Atractylodes macrocephala Koidz and Its Effect on Intestinal Microflora.

An active polysaccharide from the rhizome of Atractylodes macrocephala Koidz (PAM) was identified to improve and adjust disordered intestinal flora. High-performance gel permeation chromatography (HPGPC) and gas chromatography-mass spectrometry (GC-MS) were employed to identify the components of PAM as rhamnose, glucose, mannose, xylose, and galactose at a ratio of 0.03 : 0.25 : 0.15 : 0.41 : 0.15. PAM metabolized in gastrointestinal tract when incubated with artificial gastric and intestinal juices. Anaerobic incubation of PAM on intestinal flora confirmed that PAM promoted the ability of intestinal bacteria to digest reducing sugar. Based on the Shannon index and similarity coefficient index of enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting of the total intestinal bacteria DNA, we concluded that PAM can significantly improve disordered intestinal flora and may be used as an oral adjuvant to regulate intestinal flora.

[Effects of polymer aluminum hydrolysis on phosphorus distribution in coagulation].

Coagulation treatment was conducted by using coagulants of different basicity (ratio of OH(-)/Al); contents,distributions and algal availability of phosphorus in water were studied before and after coagulation. Results show that: phosphorus removals and its distribution in water were markedly different according to the coagulant with different basicity used; Al(a) plays an important role in the coagulation experiment for P removal. The lower the coagulant basicity was, the higher phosphorus removal was achieved; and PACl0 showed the best performance. Dissolved and particulate phosphorus reduced gradually with the increase of the coagulant (PACl0). They were entirely turned into deposit phosphorus when the coagulant dosage was above 10 mg x L(-1). The demand of coagulant for turbidity control was proved to be unequal to that for phosphorus removal. The coagulant dosages of about 3-5 mg x L(-1) achieved the best turbidity removal in the experiment; while much higher dosage was needed to get desired phosphorus removal. The amount of AAP (algal available phosphorus) in the sediments changed according to coagulant (PAC10) dosages. AAP increased with the increase of coagulant dosage when the dosage was less than 5 mg x L(-1), then it decreased with further addition of coagulant above 5 mg x L(-1). It was proved that release of phosphorus in sediments would be controlled effectively by addition of coagulant overdosed compared to the need for turbidity removal, which is important to long-term control of phosphorus. It was indicated that the dosage of coagulant used for phosphorus removal can not use the sole criterion for turbidity removal; the need for total phosphorus removal, sediment release of available phosphorus (such as AAP) and other phosphorus control requirements should be considered; and a larger dosage would be needed.