This study establishes a high-performance liquid chromatography (HPLC) method for the determination of acetate content in hemodialysis solutions and dialysis concentrates. In this study, Synergi Polar-RP column is utilized. Phosphate buffered saline (50 mmol/L, pH=2.5) is used as a mobile phase. The flow rate is 1.0 mL/min. The wavelength of detection is 212 nm. Results show that the linear relationship of acetate is good in the range of 0.1~20 mmol/L, r =0.999 9 and the spike recoveries are from 98.9%~99.5%, RSD<0.5% ( n=3). This method can easily and accurately determine the acetate content in hemodialysis solutions and dialysis concentrates, and can be applied to quality control in the production and use of such products.
Chromatography, High Pressure Liquid/methods* ; Acetates/analysis* ; Hemodialysis Solutions/analysis* ; Dialysis Solutions/analysis* ; Renal Dialysis

OBJECTIVES: To explore the mechanism of Lacticaseibacillus paracasei E6 for improving vinorelbine-induced immunosuppression in zebrafish. METHODS: The intestinal colonization of L. paracasei E6 labeled by fluorescein isothiocyanate (FITC) in zebrafish was observed under fluorescence microscope. In a zebrafish model of vinorelbine-induced immunosuppression, the immunomodulatory activity of L. paracasei E6 was assessed by analyzing macrophage and neutrophil counts in the caudal hematopoietic tissue (CHT), the number of T-lymphocyte, and the expressions of interleukin-12 (IL-12) and interferon-γ (IFN-γ). The contents of short-chain fatty acids (SCFAs) in L. paracasei E6 fermentation supernatant and the metabolites of L. paracasei E6 in zebrafish were detected by LC-MS/MS-based targeted metabolomics. The immunomodulatory effects of the SCFAs including sodium acetate, sodium propionate and sodium butyrate were evaluated in the zebrafish model of immunosuppression. RESULTS: After inoculation, green fluorescence of FITC-labeled L. paracasei E6 was clearly observed in the intestinal ball, midgut and posterior gut regions of zebrafish. In the immunocompromised zebrafish model, L. paracasei E6 significantly alleviated the reduction of macrophage and neutrophil counts in the CHT, increased the fluorescence intensity of T-lymphocytes, and promoted the expressions of IL-12 and IFN-γ. Compared with MRS medium, L. paracasei E6 fermentation supernatant showed significantly higher levels of acetic acid, propionic acid and butyric acid, which were also detected in immunocompromised zebrafish following treatment with L. paracasei E6. Treatment of the zebrafish model with sodium acetate and sodium propionate significantly increased macrophage and neutrophil counts in the CHT and effectively inhibited vinorelbine-induced reduction of thymus T cells. CONCLUSIONS L. paracasei E6 can improve vinorelbine-induced immunosuppression in zebrafish through its SCFA metabolites acetic acid and propionic acid.
Animals ; Zebrafish/immunology* ; Acetic Acid/metabolism* ; Propionates/metabolism* ; Fatty Acids, Volatile/metabolism*

OBJECTIVE: To explore the mechanism of antibiotic delivery system targeting bacterial biofilm with linezolid (LZD) based on ε-poly- L-lysine (ε-PLL) and cyclodextrin (CD) (ε-PLL-CD-LZD), aiming to enhance antibiotic bioavailability, effectively penetrate and disrupt biofilm structures, and thereby improve the treatment of bone and joint infections. METHODS: ε-PLL-CD-LZD was synthesized via chemical methods. The grafting rate of CD was characterized using nuclear magnetic resonance. In vitro biocompatibility was evaluated through live/dead cell staining after co-culturing with mouse embryonic osteoblast precursor cells (MC3T3-E1), human umbilical vein endothelial cells, and mouse embryonic fibroblast cells (3T3-L1). The biofilm-enrichment capacity of ε-PLL-CD-LZD was assessed using Staphylococcus aureus biofilms through enrichment studies. Its biofilm eradication efficacy was investigated via minimum inhibitory concentration (MIC) determination, scanning electron microscopy, and live/dead bacterial staining. A bone and joint infection model in male Sprague-Dawley rats was established to validate the antibacterial effects of ε-PLL-CD-LZD. RESULTS: In ε-PLL-CD-LZD, the average grafting rate of CD reached 9.88%. The cell viability exceeded 90% after co-culturing with three types cells. The strong biofilm enrichment capability was observed with a MIC of 2 mg/L. Scanning electron microscopy observations revealed the effective disruption of biofilm structure, indicating potent biofilm eradication capacity. In vivo rat experiments demonstrated that ε-PLL-CD-LZD significantly reduced bacterial load and infection positivity rate at the lesion site ( P<0.05). CONCLUSION The ε-PLL-CD antibiotic delivery system provides a treatment strategy for bone and joint infections with high clinical translational significance. By effectively enhancing antibiotic bioavailability, penetrating, and disrupting biofilms, it demonstrated significant anti-infection effects in animal models.
Biofilms/drug effects* ; Animals ; Anti-Bacterial Agents/pharmacology* ; Polylysine/chemistry* ; Cyclodextrins/administration & dosage* ; Humans ; Linezolid/pharmacology* ; Staphylococcus aureus/physiology* ; Rats, Sprague-Dawley ; Mice ; Rats ; Male ; Drug Delivery Systems ; Staphylococcal Infections/drug therapy* ; Microbial Sensitivity Tests ; Human Umbilical Vein Endothelial Cells ; Osteoblasts/cytology*

OBJECTIVE: The present study evaluated the effects of deep acupuncture at Weizhong acupoint (BL40) on bladder function and brain activity in a rat model of overactive bladder (OAB), and investigated the possible mechanisms around the acupuncture area that initiate the effects of acupuncture. METHODS: Adult female Sprague-Dawley rats were randomly divided into six groups, comprising a control group, model group, group treated with deep acupuncture at BL40, group treated with shallow acupuncture at BL40, group treated with acupuncture at non-acupoint next to BL40, and group treated with acupuncture at Xuanzhong (GB39). Urodynamic evaluation was used to observe the urination, and functional magnetic resonance imaging was used to observe the brain activation. The mechanism of acupuncture at BL40 in regulating bladder function was explored by toluidine blue staining and enzyme-linked immunosorbent assay, and the mechanism was verified by stabilizing mast cells (MCs) or blocking tibial nerve. RESULTS: Deep acupuncture at BL40 significantly increased the intercontraction interval in OAB rats and enhanced the mean amplitude of low frequency fluctuation of primary motor cortex (M1), periaquaductal gray matter (PAG), and pontine micturition center (PMC). It also increased the zero-lag functional connectivity between M1 and PAG and between PAG and PMC. Shallow acupuncture at BL40 and acupuncture at non-acupoint or GB39 had no effect on these indexes. Further studies suggested that deep acupuncture at BL40 increased the number and degranulation rate of MCs as well as the contents of 5-hydroxytryptamine, substance P, and histamine in the tissues around BL40. Blocking the tibial nerve by lidocaine injection or inhibiting MC degranulation by sodium cromoglycate injection obstructed the effects of acupuncture on restoring urinary function and modulating brain activation in OAB rats. CONCLUSION Deep acupuncture at BL40 may be more effective for inhibiting OAB by promoting degranulation of MCs around the acupoint and stimulating tibial nerve, thereby regulating the activation of the brain area that controls the lower urinary tract. Please cite this article as: Liu X, Zhang CY, Du XY, Li SS, Wang YQ, Zheng Y, Deng HZ, Fang XQ, Li JY, Wang ZQ, Xu SF, Mi YQ. Acupuncture at Weizhong (BL40) attenuates acetic acid-induced overactive bladder in rats by regulating brain neural activity through the modulation of mast cells and tibial nerves. J Integr Med. 2025; 23(1): 46-55.
Animals ; Urinary Bladder, Overactive/physiopathology* ; Mast Cells/physiology* ; Rats, Sprague-Dawley ; Female ; Acupuncture Therapy ; Acupuncture Points ; Rats ; Brain/physiopathology* ; Tibial Nerve/physiopathology* ; Acetic Acid ; Urinary Bladder/physiopathology*

Anthoceros angustus Steph. is rich in phenolic acids such as rosmarinic acid (RA). Phenylalanine ammonia-lyase (PAL) is an entry enzyme in the phenylpropanoid pathway of plants, playing an important role in the biosynthesis of RA. To investigate the important role of PAL in rosmarinic acid synthesis, two PAL genes (designated as AanPAL1 and AanPAL2) were cloned from A. angustus, encoding 755 and 753 amino acid residues, respectively. The AanPAL deduced amino acid sequences contain the conserved domains of PAL and the core active amino acid residues Ala-Ser-Gly. The phylogenetic analysis indicated that AanPAL1 and AanPAL2 were clustered with PALs from bryophytes and ferns and had the shortest evolutionary distance with the PALs from Physcomitrella patens. Quantitative real-time PCR results showed that the expression of AanPAL1 and AanPAL2 was induced by exogenous methyl jasmonate (MeJA). HPLC results showed that the MeJA treatment significantly increased the accumulation of RA. AanPAL1 and AanPAL2 were expressed in Escherichia coli and purified by histidine-tag affinity chromatography. The recombinant proteins catalyzed the conversion of L-phenylalanine to generate trans-cinnamic acid with high efficiency, with the best performance at 50 ℃ and pH 8.0. The K_m and k_cat of AanPAL1 were 0.062 mmol/L and 4.35 s^-1, and those of AanPAL2 were 0.198 mmol/L and 14.48 s^-1, respectively. The specific activities of AanPAL1 and AanPAL2 were 2.61 U/mg and 8.76 U/mg, respectively. The two enzymes had relatively poor thermostability but good pH stability. The high activity of AanPAL2 was further confirmed via whole-cell catalysis with recombinant E. coli, which could convert 1 g/L L-phenylalanine into trans-cinnamic acid with a yield of 100% within 10 h. These results give insights into the regulatory role of AanPAL in the biosynthesis of RA in A. angustus and provide candidate enzymes for the biosynthesis of cinnamic acid.
Phenylalanine Ammonia-Lyase/metabolism* ; Cloning, Molecular ; Cinnamates/metabolism* ; Recombinant Proteins/metabolism* ; Rosmarinic Acid ; Depsides/metabolism* ; Escherichia coli/metabolism* ; Amino Acid Sequence ; Plant Proteins/metabolism* ; Phylogeny ; Acetates/pharmacology* ; Cyclopentanes ; Oxylipins

This study aimed to investigate the toxicity differences of the ethyl acetate fraction of Euphorbiae Pekinensis Radix before and after vinegar processing and explore the detoxification mechanism of vinegar processing using non-targeted metabolomics. The changes in terpenoid components in the ethyl acetate fraction before and after vinegar processing were analyzed using UFLC-Q-TOF-MS. Normal rats were orally administered the raw and vinegar-processed ethyl acetate fractions of Euphorbia Pekinensis Radix. The toxicity differences in ethyl acetate fractions of Euphorbia Pekinensis Radix before and after vinegar processing were evaluated by pathological morphology, serum liver and kidney function, oxidative damage, and inflammatory injury indicators, and apoptosis factors. Serum metabolomics technology was utilized to identify changes in endogenous metabolites. Principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) were employed to identify differential metabolites and metabolic pathways related to the detoxification of vinegar-processed Euphorbia Pekinensis Radix. The content of terpenoid components in the ethyl acetate fraction of Euphorbia Pekinensis Radix significantly decreased after vinegar processing. Histopathological sections and various indicators revealed that both the raw and vinegar-processed ethyl acetate fractions of Euphorbia Pekinensis Radix could induce toxicity in the liver, stomach, and intestine, with a reduction after vinegar processing. The toxicity was associated with oxidative damage, inflammatory injury, and apoptosis. A total of 13 differential metabolites and 5 main metabolic pathways related to Euphorbia Pekinensis Radix toxicity were identified by serum metabolomics. PCA and PLS-DA score plots indicated that both the raw and vinegar-processed ethyl acetate fractions disrupted the endogenous metabolic profiles in rats, mainly concentrating on lipid metabolism, primary bile acid biosynthesis, and arachidonic acid metabolism, with vinegar processing alleviating these metabolic disruptions. Therefore, the ethyl acetate fraction of Euphorbia Pekinensis Radix possesses liver, stomach, and intestinal toxicity, and vinegar processing reduces its toxicity by decreasing the content of terpenoid components. The detoxification mechanism may be related to alleviating oxidative damage, inflammatory injury, apoptosis, and improving lipid metabolism.
Euphorbia/chemistry* ; Animals ; Acetic Acid ; Metabolomics ; Rats ; Acetates/chemistry* ; Male ; Drugs, Chinese Herbal/chemistry* ; Rats, Sprague-Dawley ; Liver/metabolism*

To explore the mechanism by which vinegar-processed Euphorbiae Pekinensis Radix regulates gut microbiota and reduces intestinal toxicity, this study aimed to identify key microbial communities related to vinegar-induced detoxification and verify their functions. Using a derivatization method, the study measured the content of short-chain fatty acids(SCFAs) in feces before and after vinegar-processing of Euphorbiae Pekinensis Radix. Combined with the results of previous gut microbiota sequencing, correlation analysis was used to identify key microbial communities related to SCFAs content. Through single-bacterium transplantation experiments, the role of key microbial communities in regulating SCFAs metabolism and alleviating the intestinal toxicity of Euphorbiae Pekinensis Radix was clarified. Fecal extracts were then added to a co-culture system of Caco-2 and RAW264.7 cells, and toxicity differences were evaluated using intestinal tight junction proteins and inflammatory factors as indicators. Additionally, the application of a SCFAs receptor blocker helped confirm the role of SCFAs in reducing intestinal toxicity during vinegar-processing of Euphorbiae Pekinensis Radix. The results of this study indicated that vinegar-processing of Euphorbiae Pekinensis Radix improved the decline in SCFAs content caused by the raw material. Correlation analysis revealed that Bifidobacterium was positively correlated with the levels of acetic acid, propionic acid, isobutyric acid, n-butyric acid, isovaleric acid, and n-valeric acid. RESULTS:: from single-bacterium transplantation experiments demonstrated that Bifidobacterium could mitigate the reduction in SCFAs content induced by raw Euphorbiae Pekinensis Radix, enhance the expression of tight junction proteins, and reduce intestinal inflammation. Similarly, cell experiment results confirmed that fecal extracts from Bifidobacterium-transplanted mice alleviated inflammation and increased the expression of tight junction proteins in intestinal epithelial cells. The use of the free fatty acid receptor-2 inhibitor GLPG0974 verified that this improvement effect was related to the SCFAs pathway. This study demonstrates that Bifidobacterium is the key microbial community responsible for reducing intestinal toxicity in vinegar-processed Euphorbiae Pekinensis Radix. Vinegar-processing increases the abundance of Bifidobacterium, elevates the intestinal SCFAs content, inhibits intestinal inflammation, and enhances the expression of tight junction proteins, thereby improving the intestinal toxicity of Euphorbiae Pekinensis Radix.
Animals ; Mice ; Humans ; Acetic Acid/chemistry* ; Gastrointestinal Microbiome/drug effects* ; Fatty Acids, Volatile/metabolism* ; Bifidobacterium/genetics* ; Caco-2 Cells ; Intestines/microbiology* ; Drugs, Chinese Herbal/chemistry* ; Euphorbia/toxicity* ; RAW 264.7 Cells ; Male ; Feces/chemistry* ; Intestinal Mucosa/drug effects*

Gelsemium elegans, a vine plant of Loganiaceae, has both medicinal and forage values. However, it is susceptible to low temperatures during growth. Exploring low temperature response genes is of great significance for cold resistance breeding of G. elegans. Ethylene response factors (ERFs) are the transcription factors of the AP2/ERF superfamily and play a crucial role in plant stress response. In this study, based on the unigene GeERF involved in the response to low temperature stress in the transcriptome of G. elegans, a full-length cDNA sequence of the transcription factor GeERF4B-1 was cloned from the leaves of G. elegans by reverse transcription-polymerase chain reaction (RT-PCR). Bioinformatics analysis showed that GeERF4B-1 had an open reading frame of 759 bp, encoding a protein composed of 252 amino acid residues and with a relative molecular weight of 27 kDa. The deduced protein was predicted to be an unstable, alkaline, and hydrophilic protein. The phylogenetic tree showed that GeERF4B-1 was in the same clade as the B-4 subfamily of the ERF family. The results of the subcellular localization experiment revealed that GeERF4B-1 was located in the nucleus. Real time quantitative PCR (RT-qPCR) analysis indicated that GeERF4B-1 was expressed in the root, stem, and leaf of G. elegans, with the highest expression level in the root. Compared with the control, the treatments with a low temperature (4 ℃), methyl jasmonate (MeJA), and abscisic acid (ABA) up-regulated the expression level of GeERF4B-1, which reached the peak at 24-48 h. This result revealed that GeERF4B-1 actively responded to low temperature, MeJA, and ABA stresses. However, both sodium chloride (NaCl) and drought treatments down-regulated the expression of GeERF4B-1. In addition, a prokaryotic expression vector of GeERF4B-1 was constructed, and a fusion protein of approximately 52 kDa was yielded after induced expression. The results of the plate stress assay showed that compared with the control, the prokaryotic strain expressing GeERF4B-1 demonstrated enhanced tolerance to low temperatures and sensitivity to salt and mannitol stresses. This study provides theoretical references and potential genetic resources for breeding G. elegans varieties with stress resistance.
Transcription Factors/metabolism* ; Plant Proteins/metabolism* ; Gelsemium/metabolism* ; Acetates/pharmacology* ; Gene Expression Regulation, Plant ; Phylogeny ; Cold Temperature ; Amino Acid Sequence ; Cyclopentanes/metabolism* ; Oxylipins/metabolism* ; Stress, Physiological/genetics* ; Abscisic Acid/metabolism* ; Cloning, Molecular

OBJECTIVE: To observe the effects of electroacupuncture at "Siguan" points on behavior, colonic 5-hydroxytryptamine (5-HT) and fecal short-chain fatty acids (SCFAs) in rats with post-stroke depression (PSD), and explore the effect mechanism of electroacupuncture at Siguan points on PSD. METHODS: Fifty SD rats were randomly divided into a sham-operation group, a stroke group, a PSD group, a drug group and an electroacupuncture group, with 10 rats in each one. The stroke model was established by middle cerebral artery occlusion (MCAO) method in the stroke group; except for the sham-operation group, the rats in the other groups were intervened with MCAO combined with solitary and chronic unpredictable mild stress (CUMS) to establish PSD model. In the electroacupuncture group, electroacupuncture was delivered at "Hegu" (LI 4) and "Taichong" (LR 3), with disperse-dense wave, 2 Hz/10 Hz in frequency, for 30 min in each intervention, once daily, for consecutive 21 days. Simultaneously, distilled water (0.01 L•kg^-1•d^-1) was administrated intragastrically. Fluoxetine solution (2.33 mg•kg^-1•d^-1) was given by gavage , once a day and for 21 days in the drug group. The same procedure of fixation and gavage with distilled water were adopted in the sham-operation group, the stroke group and the PSD group. Separately, before stroke modeling, after PSD modeling and after 21-day intervention, the consumption of sugar water and the scores of horizontal movement and vertical movement in open-field test were observed. After 21-day intervention, the content of colonic 5-HT was detected by immunohistochemical method, and that of fecal SCFAs was determined by gas chromatography mass spectrometry. RESULTS: After PSD modeling, compared with the stroke group, the sugar water consumption, the horizontal movement scores and vertical movement scores of the open-field test were all reduced in the PSD group, the drug group and the electroacupuncture group (P<0.05). After 21-day intervention, the sugar water consumption and the scores of horizontal movement and vertical movement of the open-field test were increased in the drug group and the electroacupuncture group (P<0.05) when compared with the PSD group; and the horizontal movement score in the electroacupuncture group was lower than that of the drug group (P<0.05). Compared with the sham-operation group, the contents of total fecal SCFAs and acetic acid were lower in the stroke group (P<0.05), and the contents of colonic 5-HT and total fecal SCFAs, acetic acid, propionic acid and butyric acid were reduced in the PSD group (P<0.05). In comparison with the PSD group, the contents of colonic 5-HT and total fecal SCFAs, acetic acid and propionic acid were increased in the drug group and the electroacupuncture group (P<0.05); and the content of colonic 5-HT in the electroacupuncture group was lower than that of the drug group (P<0.05). The level of colonic 5-HT was positively correlated with the contents of total fecal SCFAs and propionic acid (r=0.424, P=0.005; r=0.427, P=0.004). CONCLUSION Electroacupuncture at "Siguan" points can relieve the depression-like behavior of PSD rats, and its underlying mechanism may be related to the regulation of fecal SCFAs, which affects the release of colonic 5-HT.
Animals ; Rats ; Rats, Sprague-Dawley ; Propionates ; Serotonin ; Depression/therapy* ; Electroacupuncture ; Fatty Acids, Volatile ; Stroke/complications* ; Acetic Acid ; Butyric Acid ; Water

This study aims to optimize the parameters for stir-frying of Kansui Radix with vinegar based on the conversion of representative toxic diterpenes, which is expected to serve as a reference for the standardized production of Kansui Radix stir-fried with vinegar. To be specific, the toxic components [3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol(3-O-EZ), kansuiphorin C(KPC)] in Kansui Radix and the products(ingenol, 20-deoxyingenol) after the stir-frying with vinegar were selected. The toxicity to intestine and water-draining activity were evaluated with NCM460(normal human colon mucosal epithelial cell line) and HT-29(a human colorectal adenocarcinoma cell line). An HPLC method was then developed to assess the conversion of toxic components. On this basis, temperature, time, and amount of vinegar for the processing of Kansui Radix were optimized with the Box-Behnken design and the content of ingenol and 20-deoxyingenol as evaluation index. The results showed that after the stir-frying of Kansui Radix with vinegar, 3-O-EZ and KPC were first converted to monoester 3-O-(2'E,4'Z-decadienoyl)ingenol(3-EZ) and 5-O-benzoyl-20-deoxyingenol(5-O-Ben) and finally to almost non-toxic ingenol and 20-deoxyingenol, respectively. Meanwhile, the water-draining activity was retained. Six compounds had a good linear relationship with the peak area in the corresponding concentration ranges(R~2≥0.999 8), and the average recovery fell in the range of 98.20%-102.3%(RSD≤2.4%). The content of representative diterpenes and intermediate products was 14.78%-24.67% lower in the Kansui Radix stir-fried with vinegar than in the Kansui Radix, while the content of the conversed products was 14.37%-71.37% higher. Among the process parameters, temperature had significant influence on the total content of products, followed by time. The optimal parameters were 210 ℃, 15 min, and 30% vinegar. The relative error between the experimental results and the predicted values was 1.68%, indicating that the process was stable and reproducible. The strategy of screening optimal parameters for stir-frying of Kansui Radix with vinegar based on the transformation of toxic components can help improve the production stability, reduce the toxicity, and ensure the efficacy of Kansui Radix stir-fried with vinegar, which can serve as a reference for the process optimization of similar toxic Chinese medicinals.
Humans ; Acetic Acid ; Euphorbia ; HT29 Cells