1.Pre-L-methionine feeding strategy for S-adenosyl-L-methionine fermentative production.
Chinese Journal of Biotechnology 2008;24(10):1824-1827
The yield of S-adenosyl-L-methionine (SAM) on high-cell-density fermentation by saccharomyces cerevisiae is mostly affected by the feeding strategy of pre-L-methionine. The mutant strain SAM0801 that could accumulate more SAM was used in this study. Six high-cell-density fermentation experiments in 5 L fermentor were investigated to get the optimal feeding time and amount of L-methionine. The results showed that when 40 g L-methionine was added in the fermentor after 30 h fermentation, a dry cell weight of 100 g/L was achieved. Under this condition, after 58 h fermentation, both the dry cell weight and the yield of SAM reached the maximum, 168 g/L and 14.48 g/L respectively.
Bioreactors
;
microbiology
;
Fermentation
;
Methionine
;
analysis
;
metabolism
;
Mutation
;
S-Adenosylmethionine
;
biosynthesis
;
Saccharomyces cerevisiae
;
genetics
;
growth & development
;
metabolism
2.Optimization of high-cell-density fermentation process for S-adenosyl-L-methionine production.
Jiepeng WANG ; Jinjun HAN ; Xiaonan LI ; Peiyi LIU ; Tianwei TAN
Chinese Journal of Biotechnology 2009;25(4):533-536
Poor stability existed in the anaphase of the high-cell-density fermentation of Saccharomyces crevisiae for S-adenosyl-L-methionine (SAM) production in 5 L fermentor. To improve the fermentation stability, we studied the addition of diammonium hydrogen phosphate, sodium glutamate and adenosine disodium triphosphate into glucose feeding solution. Study of four fed-batch cultures showed that, after 34 h fermentation, when dry cell weight reached 100 g/L, the addition of 50 g pre-L-methionine and glucose feeding with 10 g/L adenosine disodium triphosphate was optimal for SAM production. Under this condition, after 65.7 h fermentation, both the dry cell weight and the yield of SAM reached the maximum, 180 g/L and 17.1 g/L respectively.
Adenosine Triphosphate
;
pharmacology
;
Fermentation
;
Phosphates
;
pharmacology
;
S-Adenosylmethionine
;
biosynthesis
;
genetics
;
Saccharomyces cerevisiae
;
enzymology
;
genetics
;
Sodium Glutamate
;
pharmacology
3.Improvement of Lung Function in Rats with Idiopathic Pulmonary Fibrosis by Shengxiantang via Regulating Cell Senescence Mediated by Wnt3a/β-catenin Signaling Pathway
Yanan YAN ; Yulei LIANG ; Jiepeng WANG ; Chaoyi FANG
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(12):31-38
ObjectiveTo observe the effect of Shengxiantang (SXT) on cell senescence mediated by wingless/integrated (Wnt)3a/β-catenin pathway in rats with idiopathic pulmonary fibrosis (IPF) and reveal the possible mechanism in improving lung function of IPF rats. MethodA total of 32 SPF level SD rats were randomly divided into sham group, model group, pirfenidone group, and SXT group. The IPF rat model was established by intratracheal instillation of bleomycin (0.005 g·kg-1). The following day after surgery, rats in the SXT group were given the aqueous solution of SXT granules (0.78 g·kg-1), and the pirfenidone group was given pirfenidone suspension (0.05 g·kg-1). The other groups were given deionized water (10 mL·kg-1) for 28 consecutive days. Lung tissue was collected after the lung function was measured. The pathological changes of the lung tissue were observed by hematoxylin-eosin (HE) and Masson staining, and then the Szapiel score and Ashcroft score were performed. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect telomere length. Western blot was applied to detect the expressions of epithelial-mesenchymal transformation (EMT) markers [α-smooth muscle actin (α-SMA) and E-cadherin], telomere reverse transcriptase (TRET), aging-related proteins (p53 and p21), senescence-associated secretory phenotype [interleukin-6 (IL-6) and matrix metalloproteinase-1 (MMP-1)], and key proteins of Wnt signaling pathway [Wnt3a, glycogen synthase kinase-3β (GSK-3β), β-catenin, Cyclin D1, and c-Myc]. ResultCompared with those in the Sham group, peak expiratory flow (PEF) and minute ventilation volume (MV) in the model group were significantly decreased (P<0.01), and the frequency of respiratory (f) was significantly increased (P<0.01). The Szapiel score, Ashcroft score, and protein expression of α-SMA, p53, p21, IL-6, MMP-1, Wnt3a, GSK3β, β-catenin, Cyclin D1, and c-Myc were increased (P<0.01). The expressions of E-cadherin and TERT, as well as telomere length were significantly decreased (P<0.01). Compared with those in the model group, PEF and MV in the SXT group were significantly increased (P<0.01), while f was significantly decreased (P<0.01). The Szapiel score, Ashcroft score, and protein expression of α-SMA, p53, p21, IL-6, MMP-1, Wnt3a, GSK3β, β-catenin, Cyclin D1, and c-Myc were significantly decreased (P<0.05, P<0.01). Nevertheless, the expression of E-cadherin and TERT, as well as telomere length were significantly increased (P<0.01). ConclusionSXT presents a significant protective effect on lung function in IPF rats, and the prescription may act on the Wnt3a/β-catenin signaling pathway to regulate cell senescence induced by TERT to inhibit EMT.
4.Effect of Yupingfeng Granules (玉屏风颗粒) on Metabonomics of Alveolar Lavage Fluid and Gut Microflora in Chronic Obstructive Pulmonary Disease Model Rats
Haiye LIU ; Shan LUO ; Zhiling JIA ; Wenjun DONG ; Yuanyuan WANG ; Jiepeng WANG ; Wentao YU
Journal of Traditional Chinese Medicine 2023;64(20):2116-2124
ObjectiveTo explore the possible mechanism of Yupingfeng Granules (玉屏风散) in preventing and treating chronic obstructive pulmonary disease (COPD) from the perspective of “lung-gut axis”. MethodsThirty-two male Wistar rats were randomly divided into normal group,model group, roxithromycin group and Yupingfeng Granules group, with 8 rats in each group. Except for the normal group, the rat model of COPD was prepared by intratracheal instillation of lipopolysaccharide (LPS) combined with smoking for 12 weeks. Since the fifth week of modeling,the roxithromycin group and the Yupingfeng Granules group were given 31.5 mg/(kg·d) and 1.575 g/(kg·d) of corresponding drugs respectively by gavage,and normal group and model group were given 10 ml/(kg·d) physiolo-gical saline. Sample was collected 24 hours after the last administration. The pathological changes of lung tissue were observed using HE staining; Ultrahigh performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOFMS) was used to detect the differential metabolites in alveolar lavage fluid (BALF) in all groups but roxithromycin group;16S rDNA sequencing technology was used to detect the changes of intestinal flora, and the association analysis was conducted between the differential metabolites and the differential flora. ResultsCompared with the normal group, the model group showed an increase in goblet cells in the small bronchial wall, disappearance of the smooth muscle layer of the bronchial wall, and infiltration of inflammatory cells; compared with the model group, roxithromycin group showed slight alveolar interstital edema, and obviously reduced inflammatory cell, while no obvious alveolar interstital edema was observed in the Yupingfeng Granules group, showing a small amout of inflammatory cell infiltration. The results of the BALF differential metabolite screening showed that compared with the normal group, 12 substances were upregulated and 19 substances were downregulated in the model group; compared with the model group, 37 substances in the Yupingfeng Granules group were upregulated and 43 substances were downregulated KEGG analysis yielded a total of 2 metabolic pathways, glycerophospholipid metabolism, and unsaturated fatty acid biosynthetic metabolism; compared with the model group, choline, acetylcholine, glycerol-3-phosphate, glycerophosphate choline, palmitic acid, and arachidonic acid showed an upward trend, while stearic acid and docosahexaenoic acid showed a downward trend in Yupingfeng Granules group (P<0.05). The results of the intestinal flora showed that, there are 80 different species between the normal group and the model group, and 65 different species between the model group and Yupingfeng Granules group. Among the top 5 species with relative abundance levels,compared with the model group, the level of Prevotella_9,Ruminococcaceae_UCG-005,Ruminiclostridium_6 increase,and Lactobacillus,Bacteroides decrease(P<0.05).The results of the correlation analysis showed that, in the normal and model groups, arachidonic acid was negatively correlated with Oribacterium(r=
5.Effect of Buyang Huanwutang on Keap1/Nrf2/HO-1 Antioxidant Signaling Pathway in Rats with Idiopathic Pulmonary Fibrosis
Zhe ZHANG ; Shu ZHAO ; Yunpeng HAN ; Feng JIN ; Jiarong SHANG ; Jiepeng WANG ; Chaoyi FANG
Chinese Journal of Experimental Traditional Medical Formulae 2022;28(17):9-16
ObjectiveTo study the effect of Buyang Huanwutang on Kelch-like Ech-related protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant signaling pathway in rats with idiopathic pulmonary fibrosis (IPF) and explore the mechanism of this prescription in the treatment of IPF. MethodForty SPF-grade male SD rats were assigned into a sham operation group, a model group, a Buyang Huanwutang group, and a nintedanib group according to random number table method, with 10 rats in each group. IPF rat model was established by intratracheal infusion of bleomycin (0.005 g·kg-1) in other groups except the sham operation group. Buyang Huanwutang group was administrated with Buyang Huanwutang (14.84 g·kg-1),intragastric administration of nitedanib suspension (0.1 g·kg-1),sham operation group and model group were given equal volume of normal saline, for 28 days. After lung function test, serum and lung tissue samples were collected. Hematoxylin-eosin (HE) staining and Masson trichrome staining were employed to observe the pathological changes of the lung tissue. The content of hydroxyproline (HYP) in lung tissue was detected. The levels of malondialdehyde (MDA) in serum and lung tissue, and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were determined. The mRNA and protein levels of Keap1, Nrf2, and HO-1 was determined by Real-time fluorescent quantitative polymerase chain reaction, immunohistochemical staining, and Western blot. ResultCompared with the sham operation group, the modeling increased the resistance and elasticity and decreased the compliance of respiratory system (P<0.01), elevated the lung index, pathological score, and HYP content in lung tissue (P<0.01), and enriched MDA in serum and lung tissue, while it decreased the activities of SOD, GSH-Px, and CAT (P<0.01). Furthermore, the modeling down-regulated the mRNA and protein levels of Keap1 and up-regulated those of Nrf2 and HO-1 in lung tissue (P<0.01). Compared with the model group, Buyang Huanwutang decreased the resistance and elasticity and increased the compliance of respiratory system (P<0.01), lowered the lung index, pathological score, and HYP content in lung tissue (P<0.01), and reduced MDA in serum and lung tissue, while it increased the activities of SOD, GSH-Px, and CAT (P<0.01). Additionally, Buyang Huanwutang down-regulated the expression of Keap1 and up-regulated that of Nrf2 and HO-1 in lung tissue (P<0.05, P<0.01). ConclusionBuyang Huanwutang can activate Keap1/Nrf2/HO-1 signaling pathway to enhance the antioxidant capacity and slow down the pathological process of IPF in rats.
6.Mechanism of Bugansan Decoction in ameliorating learning and memory impairment in D-galactose-induced aging rats based on AGEs/RAGE/NF-κB pathway
Wei LI ; Shuo WANG ; Jiepeng WANG ; Fang FANG ; Chaoyi FANG
Digital Chinese Medicine 2023;6(3):317-327
:
Objective To investigate the underlying mechanism of the compound Bugansan Decoction
(补肝散, BGSD) in intervening learning and memory in D-galactose (D-gal)-induced aging rats.
Methods:
A total of 40 rats were randomly assigned to four groups: control, model, BGSD [14.06 g/(kg·d)], and piracetam [0.4 g/(kg·d)] groups, with 10 rats in each group. D-gal [400 mg/(kg·d)] was injected intraperitoneally to establish the aging rat model. The rats' body weight, water intake, food intake, and gripping strength were recorded each week. The eightarm maze and step-down test were used to measure the rats' capacity for learning and memory. Liver, thymus, spleen, and brain tissues were weighed to calculate the corresponding organ indices; serum malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured. Hematoxylin and eosin (HE) staining was adopted to observe the pathological changes of the hippocampus; enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in the hippocampus. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of receptors for advanced glycation end products (RAGE), nuclear factor-κB (NF-κB), TNF-α, IL-6, and IL-1β mRNA in the hippocampus. Western blot (WB) was employed to detect the expression levels of advanced glycation end products (AGEs), RAGE, and NF-κB protein in the hippocampus.
Results:
In D-gal-induced aging rats, BGSD significantly increased food intake, water intake, body weight, gripping strength, and organ indices (P < 0.05), and significantly decreased working memory error (WME), reference memory error (RME), and total memory errors (TE) in an eight-arm maze (P < 0.05). In the step-down test, step-down latency was prolonged and the frequency of errors dropped (P < 0.05). Additionally, BGSD could lessen the harm done to hippocampus neurons, increase serum SOD activity, lower MDA levels, and down-regulate the expression levels of the pro-inflammatory molecules TNF-α, IL-6, and IL-1β (P < 0.05). Further findings showed that BGSD significantly decreased hippocampal AGEs, RAGE, and NF-κB expression (P < 0.05).
Conclusion
By blocking the AGEs/RAGE/NF-κB signaling pathway, BGSD may regulate the neuroinflammatory damage in D-gal-induced aging rats, and thus improve learning and memory.