Effects of Polymannuronic Acid on the Intestinal Microbiota in Mice after Long-Term Intragastric Administration.

Polymannuronic acid (PM) is an alginate oligosaccharide derived from brown algae with a characterized structure and excellent biological activities. Herein, mice were given different doses of PM through 30-day-long-term intragastric administration, and the contents of the jejunum, ileum, and colon were analyzed by 16S rRNA gene sequencing technology for microbial diversity, and relevant experiments were verified according to the analysis results so as to comprehensively evaluate the effects of PM on the intestinal flora. The PM (400 mg/kg and 100 mg/kg) could regulate the microflora balance at the phylum level and increase the microflora richness in the jejunum, ileum, and colon of the mice. The PM could induce more strains that are negatively correlated with Escherichia, thereby reducing the relative abundance of Escherichia. Analysis of bacterial function showed that high and low doses of PM could promote lipid metabolism in the bacterial communities. Moreover, the PM could reduce serum total cholesterol and cholesterol ester levels in a concentration-dependent manner. High-dose PM could lead to colonic intestinal inflammation by increasing the relative abundance of multiple bacterial groups in the jejunum, ileum, and colon. Moreover, high-dose PM could increase lipopolysaccharide-binding protein and interleukin-1ß levels. Therefore, the dose of PM plays an important role in its efficacy, and its biological activity is dosedifferent.

Study on the regulatory effect of leech peptide HE-D on macrophages in atherosclerosis by transcriptome sequencing.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence of atherosclerotic cardiovascular disease is a serious threat to human health. Leeches are used in traditional Chinese medicine to treat cardiovascular diseases. HE-D is an active peptide extracted and isolated from leeches, which can inhibit the migration of RAW264.7 macrophages. AIM: This study shows the effects of HE-D on macrophages in atherosclerosis and the mechanism of inhibition on the migration of macrophages based on transcriptome sequencing (RNA-Seq). MATERIALS AND METHODS: The transwell method was used to detect the activity of HE-D in inhibiting the migration of macrophages. Macrophages were divided into control group, lipopolysaccharide group, and HE-D group. Samples were collected and RNA-Seq performed. The DEseq2 method detected significantly differentially expressed genes (DEGs), GO and KEGG Pathway databases were used to analyze the functions and pathway enrichment of DEGs. Finally, qRT-PCR and Western blotting were used to verify the genes screened by RNA-Seq analyses. RESULTS: Cell experiments showed that HE-D can inhibit the migration of RAW264.7 macrophages induced by LPS. DEseq2 analyses showed that there were 363 DEGs after HE-D administration in the result of RNA-Seq. The GO function of DEGs was significantly enriched in cell migration and inflammation, and the DEGs related to cell migration were significantly enriched in the NF-κB signaling pathway. qRT-PCR and Western blot analyses, showed that when compared with the LPS group, the related genes IKKα, IKKγ, TRAF6, TLR4, and TRAF5 in the NF-κB pathway were significantly down-regulated in the HE-D group. In addition, it was found that the inflammatory factors iNOS and TNF-α were significantly down-regulated, and Arg-1 and IL-10 were up-regulated. CONCLUSION: HE-D can inhibit the migration of macrophages by inhibiting IKKα and IKKγ in the NF-κB signaling pathway, and promote the transformation of macrophages from M1to M2 subtypes. Therefore, HE-D can potentially be used as a drug for the treatment of atherosclerosis.

A peptide inhibitor of macrophage migration in atherosclerosis purified from the leech Whitmania pigra.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis has become a worldwide public health problem that seriously threatens human health. Leech is traditional Chinese medicine that can be utilized to treat cardiovascular disease. Based on the anti-atherosclerosis activity of leech hydrolysate, we separated and purified the leech peptide capable of inhibiting macrophage migration and studied the pathways of the anti-migration leech peptide. MATERIALS AND METHODS: The leech peptide capable of inhibiting macrophage migration that measured by cell migration assays from the leech Whitmania pigra was separated and purified by Q Sepharose FF strong alkaline anion exchange column chromatography, Superdex 30, Superdex peptide and G10 gel column chromatography. And the purity, molecular weight of the leech peptide was determined by high-performance liquid chromatography and high-resolution mass spectrometry. The pathways of anti-migration to macrophages of the leech peptide were studied by inhibitors, Western blotting and RT-PCR. RESULTS: We obtained a purified leech peptide with a sequence of EAGSAKELEGDPVAG from the leech Whitmania pigra. We also showed that the anti-migration to macrophages of the leech peptide was blocked by c-Jun N-terminal kinase (JNK) inhibitor and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor. Moreover, the result of RT-PCR and Western blotting revealed that the leech peptide induced an increase in JNK, p38 phosphorylation and the transcription of mitogen-activated protein kinase kinase kinase 4 (MEKK4) and apoptosis signal-regulating kinase 2 (ASK2). These data indicated that the anti-migration to macrophages of the leech peptide occurred through JNK and p38 MAPK pathways. In addition, the results demonstrated that the leech peptide had no significant effect on the immunological activity of macrophages including phagocytic ability, lysozyme activity, and levels of expression of inflammatory factors. CONCLUSION: A sequence peptide was obtained from the hydrolysate of leech Whitmania pigra that inhibits macrophage migration.

Development of double network gels based on soy protein isolate and sugar beet pectin induced by thermal treatment and laccase catalysis.

Soy protein isolate (SPI) and sugar beet pectin (SBP) were adopted to fabricate double network (DN) gels via thermal treatment and laccase-catalysis. The concentrations of SBP (0.5%-2.5%, w/w) and SPI (4%-8%, w/w) were evaluated. DN gels showed higher holding water capacity (WHC, above 83%), compared with SBP single gel (75.96%). The presence of SPI improved the mechanical properties of gels significantly. Apparent phase separation could be observed when SPI concentration was 4%. Moreover, interpenetrating networks gradually formed with the increase of SPI concentration. The favorable structural heterogeneity and mechanical integrity derived from these polymers might be mainly responsible for the enhancement of the mechanical properties. The presence of SBP and laccase could improve the ß-sheet amounts of SPI and make it form more rigid structure according to the results of circular dichroism (CD) and fluorescence spectra. The excellent performance of DN gels could enable the delivery of various components.

Whitmania Pigra Whitman Extracts Inhibit Lipopolysaccharide Induced Rat Vascular Smooth Muscle Cells Migration and their Adhesion Ability to THP-1 and RAW 264.7 Cells.

AIM: Atherosclerosis is a kind of chronic inflammatory disease. A crucial pathology change of atherosclerosis is the migration of activated VSMCs to the intima where they interact with leukocytes by expressing adhesion molecules, including intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Moreover, monocyte chemoattractant protein-1 (MCP-1) expressed by VSMCs plays an important role in recruiting monocytes and macrophages. Leech (Whitmania pigra Whitman) is a traditional Chinese medicine to treat cardiovascular diseases including atherosclerosis, however previous research has rarely reported the molecular mechanism for its curative effect. Thus, our study focuses on the effects of leech extracts on the expression of inflammatory factors, adhesion molecules and MCP-1 in rat VSMCs. METHODS: In our present study, wound-healing assay and Boyden chamber model were applied to evaluate the anti-migration effect of LEE (Leech Enzyme Extracts) on LPS induced VSMCs. The anti-adhesion effect was assessed using DiI-labeled THP-1 and RAW264.7. RESULTS: LEE suppressed LPS-induced VSMCs migration and decreased the chemotaxis and adhesive capacity of THP-1 and RAW264.7 to LPS-stimulated VSMCs. LEE also attenuated the upregulation of a variety of pro-atherosclerotic factors by inhibiting the phosphorylation of p38 MAPK. LEE was also observed to prevent NF-κB p65 nuclear localization using immune-fluorescent staining. CONCLUSIONS: In conclusion, LEE suppresses LPS-induced upregulation of inflammatory factors, adhesion molecules and MCP-1 in rat VSMCs mainly via inhibiting the p38 MAPK/NF-κB pathways, thus partly uncovered LEE's molecular mechanisms for its therapeutic effect on atherosclerosis.

Effect of earthworm active protein on fibroblast proliferation and its mechanism.

CONTEXT: Earthworms have been used as a traditional medicine in China from thousands of years. In recent years, research has demonstrated that earthworm extracts might promote wound healing; however, its mechanism is still unknown. OBJECTIVE: The study investigates the mechanism and effects of earthworm active protein (EAP), on mouse embryonic fibroblast (NIH/3T3) proliferation. MATERIALS AND METHODS: The effects of earthworm active protein (EAP) in different concentrations (0, 25, 50, 100, 150, and 200 µg/mL) on NIH3T3 cell were detected by the MTT and Brdu incorporation assay (50, 100, and 150 µg/mL). The effects of EAP (37.5, 75, and 150 µg/mL) on the cell cycle were detected by flow cytometry. The cell signaling pathways of EAP-promoting NIH3T3 cell proliferation were studied by the MTT and Western blot by using different signaling pathway inhibitors. RESULTS: The results showed that EAP (50, 100, and 150 µg/mL) could promote NIH3T3 fibroblasts proliferation (36.4 ± 4.4%, 59.1 ± 4.9%, and 71.5 ± 5.7%). The mechanism of EAP promoting NIH3T3 cell proliferation should be as follows: EAP elevated cyclin D1 expression by activating MEK/ERK signaling pathway, and then promoted cell cycle from G1 to S phase, finally caused the proliferation of NIH3T3 cell. PI3K signaling pathway may be the upstream of MEK/ERK signaling pathway. DISCUSSION AND CONCLUSION: The study demonstrates that EAP is effective in promoting effects on proliferation and migration activity of NIH3T3 cell, and the proliferation activity of EAP on NIH3T3 cell may be achieved through the PI3K→Rac→PAK→MEK signaling pathway.

Icariin inhibits atherosclerosis progress in Apoe null mice by downregulating CX3CR1 in macrophage.

Horny Goat Weed is a commonly used in Chinese herbal medicine. And it is used in multiple kinds of diseases including cardiovascular diseases. Icariin is the major component isolated from Horny Goat Weed. It is reported to have lipid-lowering effect. In atherosclerosis, icariin attenuate the enhanced prothrombotic state independently of its lipid-lowering effects. However, its detail mechanism is remaining unclear. This study aimed to investigate the effect and mechanism of icariin on atherosclerosis. We performed gene expression profiling on icariin treated LPS-stimulated RAW264.7 and its control cells. Microarray analyses identified a list of genes significantly differentially expressed after icariin treated including downregulation of CX3CR1. Apoe null mice were assigned into 3 groups: control group, diet with 30 mg/kg/d icariin and diet with 60 mg/kg/d icariin. The results showed that icariin treatment significantly reduced lesion area and macrophage infiltration. Also icariin reduced CX3CR1 and CX3CL1 protein levels in the artery wall. In conclusion, icariin could be a potential anti-atherosclerosis agent by downregulating the expression of CX3CR1.

An extract from medical leech improve the function of endothelial cells in vitro and attenuates atherosclerosis in ApoE null mice by reducing macrophages in the lesions.

AIM: Medicinal leech has been widely used as a traditional Chinese medicine in cardiovascular diseases. However, its pharmaceutical effect is not fully revealed. The goal of this study was to determine whether a leech extract has the effect of anti-atherosclerosis in ApoE −/− mice and the mechanism of this effect. METHODS AND RESULTS: In vivo experiments: ApoE −/− mice fed on high-cholesterol diet were separated into 5 groups. Control group was administrated with normal water; leech extract of low dose treatment group was given a leech extract of 0.02 g/kg/d; leech extract of medium dose treatment group was given a leech extract of 0.1 g/kg/d; leech extract of high dose treatment group was given a leech extract of 0.5 g/kg/d; simvastatin group was given simvastatin of 10 mg/kg/d. Leech extract significantly reduced atherosclerotic lesions in aortic root compared with control group. And the number of macrophage in or around the atherosclerosis plaque is significantly reduced in the leech extract groups compared with control group. In vitro experiments: human endothelial cell line, EA.hy926, was induced with TNF-α to perform endothelial dysfunction. Control group: EA.hy926 cells with no special treatment; TNF-α group: EA.hy926 cells were induced by 10 ng/ml TNF-α for 6 h; leech extract only group: EA.hy926 cells were treated with 200 mg/ml leech extract only; leech extract and TNF-α group: 200 mg/ml leech extract was applied before TNF-α induction. Protein and mRNA level were detected in each group, leech extract can decrease the expression of intercellular adhesion factor (ICAM-1) and monocyte chemotactic protein (MCP-1) compared with TNF-α group. Furthermore, it showed less adhesion and migration of THP-1 cells to EA.hy926 cells in the adhesion assay and transwell assay. The NF-κB translation to nucleus was blocked by leech extract in the NF-κB translocation assay. CONCLUSIONS: Leech extract could obviously attenuate the area of atherosclerosis lesion in ApoE −/− mice. And this effect is dose dependent. The effect is mainly a result of reduced invasion of monocyte in artery walls by blocking NF-κB translocation.

[Optimization the formulation of Eisemia foetida protein burn spray by response surface methodology].

OBJECTIVE: To optimize the formulation of Eisemia foetida protein (EFP) burn spray. METHODS: A five-factor, three-level response surface method was employed; The response variable was the proliferation effect of EFP on NIH3T3 cells. RESULTS: The optimization formulation was as follows: the proportion of EFP, glycerol and mannitol was 0.91%, 1.42% and 5%, respectively; 0.02 mol/L Na2 HPO4 and 0.01 mol/L citric acid buffer system corresponding pH value was 7.0. CONCLUSION: The response surface method is reliable, efficient and suitable for optimizing the formulation of EFP burn spray.

Protective effects of alkaloid extract from Leonurus heterophyllus on cerebral ischemia reperfusion injury by middle cerebral ischemic injury (MCAO) in rats.

The neuronal damage following cerebral ischemia is a serious risk to stroke patients. The aim of this study was to investigate the neuroprotective effects of alkaloid extract from Leonurus heterophyllus (LHAE) on cerebral ischemic injury. After 24 h of reperfusion following ischemia for 2 h induced by middle cerebral artery occlusion (MCAO), some rats were intraperitoneally administered different doses of LHAE (3.6, 7.2, 14.4 mg/kg, respectively). Neurological examination was measured in all animals. Infarct volume, myeloperoxidase (MPO) activity, levels of nitrate/nitrite metabolite (NO) and apoptosis ratio of nerve fiber in brain were determined. The results showed that LHAE at 7.2 mg/kg or 14.4 mg/kg exerted significantly decreasing neurological deficit scores and reducing the infarct volume on rats with focal cerebral ischemic injury (p<0.05). At those dose, the MPO content were significantly decreased in ischemic brain as compared with model group (p<0.05). LHAE at 14.4 mg/kg significantly decreased the NO level compared with the model group (p<0.05). In addition, LHAE significantly decreased the apoptosis ratio of nerve fiber compared with the model group (p<0.05). This study suggests that LHAE may be used for treatment of ischemic stroke as a neuroprotective agent. Further studies are warranted to assess the efficacy and safety of LHAE in patients.

[Surveys on the conditions of Keshan disease and selenium levels of 15 counties in Shandong Province in 2008].

OBJECTIVE: To investigate the conditions of Keshan disease and the selenium nutritional status of residents in Keshan disease endemic area in Shandong Province. METHODS: One site from each of 15 Keshan disease endemic counties in Shandong Province was selected for the survey and 700 subjects in each site were randomly selected for the investigation. Physical examination, electrocardiogram and X-ray radiography of the subjects and the selenium contents of hair, wheat, corn and dried sweet potato were surveyed. RESULTS: A total of 10679 people were investigated in 15 counties and 315 patients were found, including 287 cases of latent type and 28 cases of chronic type, but no acute or subacute type patients were found. There were 1776 cases of abnormal electrocardiogram. Among the 461 cases checked up by radiography, the heart of 195 cases was enlarged. The selenium content of hair, wheat, corn and dried sweet potato were (0.5191 +/- 0.5538), (0.0268 +/- 0.0045), (0.0194 +/- 0.0052) and (0.0193 +/- 0.0039) mg/kg respectively. CONCLUSION: The occurrence of Keshan disease is in a stable status in Shandong Province at present. Hair selenium of residents in Keshan disease endemic area has reached an appropriate level. The selenium nutritional status of residents has improved, and the prevalence of Keshan disease is expected to be decreased in these areas.

Enhancing effect of a sea cucumber Stichopus japonicus sulfated polysaccharide on neurosphere formation in vitro.

Neural stem/progenitor cells (NSPCs) exhibit therapeutic potential in neuronal diseases. Previously, we reported that a sulfated polysaccharide (HS) from the sea cucumber Stichopus japonicus increased the proliferation of NSPCs. Since the formation of neurospheres is related with NSPCs proliferation, we investigated the mechanism leading to neurosphere formation with and without HS. The results showed that HS significantly promoted neurosphere formation in a dose-dependent manner at concentrations between 2 and 8 µg/ml. Cell cycle analysis showed that HS increased the percentage of cells in S phase by 2.8-fold, as compared with the control. On the other hand, we observed a significantly rapid aggregation of NSPCs, resulting in formation of neurospheres as early as 2 h after HS treatment. However, the aggregation was not caused by chemotactic migration of NSPCs, as evidenced by the transwell chamber assay. Furthermore, the effect of HS on NSPCs was similar to the tumor necrosis factor-α (TNF-α) that activated nuclear factor NF-κB. Thus, we demonstrated that HS was able to promote cell proliferation and aggregation of NSPCs which could lead to the formation of neurospheres, and suggested that HS can serve as an adjuvant for promoting proliferation of NSPCs and formation of neurospheres.

Proliferative effects on neural stem/progenitor cells of a sulfated polysaccharide purified from the sea cucumber Stichopus japonicus.

To test the effects of a sulfated polysaccharide, Haishen (HS) on the viability and proliferation of neural stem/progenitor cells (NSPCs), we isolated the polysaccharide from the body wall of the sea cucumber Stichopus japonicus by enzymolysis extraction, anion-exchange and gel-permeation chromatography. HS is a highly sulfated fucoidan with a molecular weight of 4.23x10(5) Da. Due to its safety being of invertebrate origin they are less likely to contain infectious agents, the effects of HS on the viability and proliferation of NSPCs in vitro were examined by MTT assay, BrdU labeling and neurosphere formation assay, respectively. Our results showed that HS alone increased NSPC viability in a dose-dependent manner. Moreover, HS acted synergistically with fibroblast growth factor-2 (FGF-2) but not epidermal growth factor (EGF) to enhance the proliferation of NSPCs. Finally, HS did not induce apoptosis of NSPCs. Our findings suggest that HS can serve as an adjuvant for promoting the proliferation of NSPCs.

[Observation on conditions and related factors of Keshan disease in Shandong province, 1990-2007].

OBJECTIVE: To observe the serial changes of condition and related factors of Keshan disease (KSD) and provide the scientific basis for disease control and further research. METHODS: The villages in KSD epidemic area of Juxian, Yishui, Pingyi, Zoucheng counties were selected. Since 1990, every five-year was counted as a stage. For the first to third stage, 3-14 year-old residents and over 14 year-old suspicious patients were selected as surveillance subjects. And in the fourth stage, all residents were selected as surveillance subjects. The same group of surveillance subjects in each stage were observed consecutively for 5 years. Surveillance contents included physical checkup, electrocardiogram (ECG) and Xray. At the same time, the selenium (Se) concentration in hair, wheat, corn and sweet potato was measured. The economic income and grain availability were also investigated. RESULTS: Totally, 14,510 cases were visited during 18 years. The incidence of KSD was 3.02% in the first stage, 2.31% in the second stage, 3.57% in the third stages and 3.65% in the fourth stage. Totally, 14,510 cases were examined by ECG, 809 cases showed the abnormal ECG and the total incidence of abnormal ECG was 5.49%. The incidence of abnormal ECG was 3.52% -5.24% from 1990 to 2004 but was 10.97%-10.91% from 2005 to 2007. 732 of hair samples, 701 of wheat samples, 615 of corn samples and 643 of sweet potato samples were collected and the Se concentration was determined by the fluorescent method. Se levels in hair samples had increased (P < 0.05) year by year but Se levels in food have not changed significantly. The economic income and grain availability had increased gradually from 535.8 yuan and 254.6 kg per person in 1990 to 2968.0 yuan and 602.0 kg per person in 2007. CONCLUSION: The condition of KSD was in a stable situation in Shandong Province. Related factors improvement should be an important environmental condition.

[The extraction, purification and assaying of Gynostemma pentaphyllum polysaccharides].

By orthogonal design, and considering extracting efficiency and cost, optimizing the extract method of Gynostemma pentaphyllum polysaccharides. We purified the crude Gynostemma pentaphyllum polysaccharides initially, and assayed the polysaccharides content of Gynostemma pentaphyllum polysccharides. The content of Gynostemma pentaphyllum polysaccharides was sigificantly higher than the predecessor. It would provide conditions for the deep exploitation of Gynostemma pentaphyllum.