OBJECTIVES: To explore the effect mechanism of electroacupuncture (EA) on functional constipation (FC) at the combined lower he-sea and front-mu points of large intestine based on enteric neuronal autophagy. METHODS: A total of 40 SPF Kunming mice were randomly divided into 5 groups (n = 8), i.e. a control group, a model group, an acupuncture group, a 3-methyl adenine (3-MA) group, and a 3-MA + acupuncture group. Except the control group, the FC model was established by gavage with compound diphenoxylate suspension for 14 days in the other 4 groups. After successful modeling, the mice of the acupuncture group and the 3-MA + acupuncture group received EA at bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37), stimulated for 30 min with disperse-dense wave, 2 Hz/15 Hz of frequency, 1 mA of intensity. EA was delivered once daily. One course of treatment was composed of 5 days and 2 courses were needed, with an interval of 2 days. An intraperitoneal injection of 3-MA (15 mg/kg) was administered 30 min before EA in the mice of the 3-MA group and the 3-MA + acupuncture group, once daily. Before and after intervention, the time of the first black stool defecation and defecation behaviors in 6 h were observed in each group. After intervention, in every group, the small intestine propulsion rate was calculated, the colon tissue morphology was observed using HE staining, the ultrastructure of enteric neuronal autophagy was observed under transmission electron microscope, and the expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1 and neuronal nuclear antigen protein (NeuN) in neurons of colonic muscularis were determined by immunohistochemistry. RESULTS: Before intervention, when compared with those in the control group, the time of the first black stool defecation was prolonged (P<0.01, P<0.05), and numbers (P<0.01), wet weight (P<0.01, P<0.05) and water content (P<0.05, P<0.01) of stool in 6 h were reduced in the model, acupuncture, 3-MA and 3-MA + acupuncture groups. After intervention, compared with those in the control group, the time of the first black stool defecation was longer (P<0.05), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were decreased in the model group. The time of the first black stool defecation was shortened (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were increased in the acupuncture group when compared with those in the model group. The time of the first black stool defecation was extended (P<0.01), and numbers (P<0.01), wet weight (P<0.01) and water content (P<0.01) of stool in 6 h were declined in the 3-MA + acupuncture group in comparison with those in the acupuncture group. All layers of colon tissue were normal and intact in each group. When compared with the control group, the small intestine propulsion rate and the average optical density (OD) values of LC3, Beclin-1 and NeuN in neurons of colonic muscularis were decreased (P<0.01), and autophagosomes were dropped in the model group. In the acupuncture group, the small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis increased (P<0.01,P<0.05), and autophagosomes were elevated when compared with those in the model group. The small intestine propulsion rate and the average OD values of NeuN, LC3 and Beclin-1 in neurons of colonic muscularis were dropped (P<0.05,P<0.01) in the 3-MA + acupuncture group in comparison with those in the acupuncture group. CONCLUSIONS Electroacupuncture may promote enteric neuronal autophagy and increase the number of neurons so that the intestinal motility can be improved and constipation symptoms can be relieved in FC mice.
Mice ; Animals ; Electroacupuncture ; Beclin-1 ; Acupuncture Points ; Constipation/therapy* ; Intestine, Small ; Autophagy ; Water

OBJECTIVES: To explore the effect of acupuncture and moxibustion on intestinal flora in the rats with diarrhea-predominant irritable bowel syndrome (IBS-D) based on 16S rDNA technique. METHODS: Ten rats were randomized from 58 SPF-grade male SD rats to be the blank group. The remained 48 rats were prepared to be IBS-D models by the modified method of acetic acid enema combined with binding tail-clip stress. Forty successfully-modeled rats were randomly divided into a model group, an acupuncture group, a moxibustion group and a western medication group, with 10 rats in each one. In the acupuncture group, the needle was inserted at bilateral "Zusanli" (ST 36) and remained for 15 min in each rat. In the moxibustion group, the suspending moxibustion was delivered at bilateral "Zusanli" (ST 36) for 15 min. The rats in the western medication group were given pinaverium bromide suspension (10 mL/kg) by intragastric administration. The above interventions were performed once daily for consecutive 14 days. The body mass and the score of fecal trait were compared before and after modeling, as well as after intervention in each group. Fecal water content, diarrhea index and colon transit time (CTT) were measured after modeling and intervention in the rats of each group separately. After intervention, the colonic morphology of rats in each group was observed, and using 16S rDNA technique, the intestinal flora was detected. RESULTS: After modeling, compared with the blank group, the body mass and CTT were reduced (P<0.01); fecal trait scores, fecal water contents and diarrhea index increased (P<0.01) in the other 4 groups. After intervention, the body mass and CTT of the rats decreased (P<0.01), and fecal trait score, fecal water content and diarrhea index increased (P<0.01) in the model group compared with those in the blank group. In the acupuncture group, the moxibustion group and the western medication group, when compared with the model group, the body mass and CTT were elevated (P<0.01), while fecal trait scores, fecal water contents and diarrhea index declined (P<0.01). Compared with the western medication group, fecal water content decreased in the acupuncture group and the moxibustion group (P<0.05), while CTT increased in the acupuncture group (P<0.01), the body mass increased and fecal trait score was dropped in the moxibustion group (P<0.05). The colonic mucosa structure was clear and complete, and there was no obvious inflammatory cell infiltration in the blank group. The mild interstitial edema of intestinal mucosa was presented with the infiltration of few inflammatory cells in the model group. There was the infiltration of few inflammatory cells in the mucosa of the acupuncture group, the moxibustion group and the western medication group. Compared with the blank group, the indexes of Richness, Chao1, ACE and Shannon decreased in the model group (P<0.05). Indexes of Richness, Chao1 and ACE increased in the acupuncture group and the moxibustion group (P<0.05), and the Richness index in the western medication group increased (P<0.05) when compared with those in the model group. The relative abundance of Bacteroidetes, Proteobacteria and Prevotella increased (P<0.05), and that of Firmicutes and Muribaculaceae decreased (P<0.05) in the model group compared with those in the blank group. When compared with the model group, the relative abundance of Bacteroidetes, Proteobacteria and Prevotella was reduced (P<0.05), while that of Firmicutes and Muribaculaceae increased (P<0.05) in the acupuncture group, the moxibustion group and the western medication group; and that of Actinobacteria and Bifidobacterium increased in the acupuncture group and the moxibustion group (P<0.05). Compared with the blank group, the relative abundance of lipopolysaccharide (LPS) biosynthesis was elevated (P<0.05), and that of folate biosynthesis, lipoic acid metabolism, zeatin biosynthesis, ubiquinone and other terpenoid quinone biosynthesis decreased (P<0.05) in the model group. The relative abundance of LPS biosynthesis was dropped (P<0.05), and that of folate biosynthesis, lipoic acid metabolism, zeatin biosynthesis, ubiquinone and other terpenoid quinone biosynthesis increased (P<0.05) in the acupuncture group, the moxibustion group and the western medication group compared with those of the model group. CONCLUSIONS Either acupuncture or moxibustion can relieve the symptoms of IBS-D and protect intestinal mucosa, which may be associated with regulating the structure of intestinal flora and promoting nutrient metabolism and biosynthesis.
Rats ; Male ; Animals ; Irritable Bowel Syndrome/therapy* ; Moxibustion/methods* ; Rats, Sprague-Dawley ; Gastrointestinal Microbiome ; Lipopolysaccharides ; Thioctic Acid ; Ubiquinone ; Zeatin ; Acupuncture Therapy ; Diarrhea/therapy* ; Terpenes ; Water ; Folic Acid ; Acupuncture Points

Swarming motility is a typical synergistic motion, in which bacteria use flagella and Type Ⅳ Pili together to move collectively on semi-solid surfaces. Swarming motility is a hot topic of research in the field of microbiology because of its close relationship with biofilm formation, fruiting bodies formation, pathogen invasion and microbial dispersal and symbiosis. A large number of studies have been conducted on bacterial swarming motility, including changes in the expression of key proteins, changes in chemical communications between bacteria as well as mechanical changes. The expression of flagellin and the level of intracellular c-di-GMP complicatedly regulates the collective behavior of bacteria in colonies, which consequently impacts the swarming motility. The unique physical properties of swarmer cells are conducive to the expansion of the whole colony. Factors such as nutrient and water content in the surrounding growth environment of bacteria also affect the ability of bacteria to swarm to different degrees. It is challenging to construct a universal model of swarming motility based on the molecular mechanisms of swarming in the future.
Bacteria ; Flagellin ; Symbiosis ; Water

Anaerobic granular sludge (AnGS), a self-immobilized aggregate containing various functional microorganisms, is considered as a promising green process for wastewater treatment. AnGS has the advantages of high volume loading rate, simple process and low excess sludge generation, thus shows great technological and economical potentials. This review systematically summarizes the recent advances of the microbial community structure and function of anaerobic granular sludge, and discusses the factors affecting the formation and stability of anaerobic granular sludge from the perspective of microbiology. Moreover, future research directions of AnGS are prospected. This review is expected to facilitate the research and engineering application of AnGS.
Sewage/chemistry* ; Waste Disposal, Fluid ; Anaerobiosis ; Microbiota ; Water Purification ; Bioreactors/microbiology*

Objective To explore the inhibitory effects and mechanisms of benzodiazepines on Helicobacter pylori (Hp).Methods The Hp international standard strain ATCC43504 was treated with benzodiazepines diazepam,midazolam,and remimazolam,respectively.The treatments with amoxicillin and clarithromycin were taken as the positive controls,and that with water for injection as the negative control.The inhibition zone of each drug was measured by the disk diffusion method.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of each drug against Hp were determined.Hp suspension was configured and treated with diazepam and midazolam,respectively.The bacterial suspension without drug added was used as the control group.The concentration of K⁺ in each bacterial suspension was measured by an automatic biochemical analyzer before drug intervention(T₀)and 1(T₁),2(T₂),3(T₃),4(T₄),5(T₅),6(T₆),and 7 h(T₇)after intervention.Hp urease was extracted and treated with 1/2 MIC diazepam,1 MIC diazepam,2 MIC diazepam,1/2 MIC midazolam,1 MIC midazolam,2 MIC midazolam,1 mg/ml acetohydroxamic acid,and water for injection,respectively.The time required for the rise from pH 6.8 to pH 7.7 in each group was determined by the phenol red coloring method.Results The inhibition zones of diazepam,midazolam,remimazolam,amoxicillin,clarithromycin,and water for injection against Hp were 52.3,42.7,6.0,72.3,60.8,and 6.0 mm,respectively.Diazepam and midazolam showed the MIC of 12.5 μg/ml and 25.0 μg/ml and the MBC of 25 μg/ml and 50 μg/ml,respectively,to Hp.The concentrations of K⁺ in the diazepam,midazolam,and control groups increased during T₁-T₇ compared with those at T₀(all P<0.01).The concentration of K⁺ in diazepam and midazolam groups during T₁-T₄ was higher than that in the control group(all P<0.01).The time of inhibiting urease activity in the 1/2 MIC diazepam,1 MIC diazepam,2 MIC diazepam,1/2 MIC midazolam,1 MIC midazolam,and 2 MIC midazolam groups was(39.86±5.11),(36.52±6.65),(38.58±4.83),(39.25±6.19),(36.36±4.61),and(35.81±6.18)min,respectively,which were shorter than that in the acetohydroxamic acid group(all P<0.01)and had no significance differences from that in the water for injection group(all P>0.05).Conclusion Diazepam and midazolam exerted inhibitory effects on Hp,which may be related to the cleavage of Hp cells rather than inhibiting urease.
Midazolam ; Helicobacter pylori ; Urease ; Clarithromycin/pharmacology* ; Benzodiazepines/pharmacology* ; Diazepam/pharmacology* ; Amoxicillin ; Water ; Anti-Bacterial Agents/pharmacology*

Alkaloids are important active ingredients occurring in many traditional Chinese medicines, and alkaloid glycosides are one of their existence forms. The introduction of saccharide units improves the water solubility of alkaloid glycosides thus presenting better biological activity.Because of the low content in plants, alkaloid glycosides have been not comprehensively studied. In this study, ultrahigh performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry(UPLC-QTOF-MS/MS) was employed to identify and analyze the alkaloid glycosides in Coptis chinensis, Phellodendron chinense, Menispermum dauricum, Sinomenium acutum, Tinospora sagittata and Stephania tetrandra. The results showed that except Tinospora sagittata, the other five herbal medicines contained alkaloid glycosides. Furthermore, the alkaloid glycosides in each herbal medicine were identified based on UV absorption spectra, quasimolecular ion peaks in MS, fragment ions information in the MS/MS, and previous literature reports. A total of 42 alkaloid glycosides were identified. More alkaloid glycosides were identified in C. chinensis and Menispermum dauricum, and eleven in C. chinensis were potential new compounds. Furthermore, the alkaloid glycosides in the water extract of C. chinensis were coarsely se-parated by macroporous adsorption resin, purified by column chromatography with D151 cation exchange resin, ODS and MCI, combined with semi-preparative high performance liquid chromatography. Two new alkaloid glycosides were obtained, and their structures were identified by mass spectrometry and NMR data as(S)-7-hydroxy-1-(p-hydroxybenzyl)-2,2-N,N-dimethyl-1,2,3,4-tetrahydroisoquinoline-6-O-β-D-glucopyranoside and(S)-N-methyltetrahydropalmatubine-9-O-β-D-glucopyranoside, respectively. This study is of great significance for enriching the information about the chemical composition and the in-depth development of C. chinensis. Meanwhile, it can provide a reference for rapid identification and isolation of alkaloid glycosides from other Chinese herbal medicines.
Glycosides/chemistry* ; Medicine, Chinese Traditional ; Tandem Mass Spectrometry/methods* ; Coptis chinensis ; Drugs, Chinese Herbal/chemistry* ; Alkaloids/analysis* ; Plant Extracts/chemistry* ; Antineoplastic Agents ; Plants, Medicinal/chemistry* ; Water ; Chromatography, High Pressure Liquid/methods* ; Coptis/chemistry*

In this study, untargeted metabolomics was conducted using the liquid chromatography-tandem mass spectrometry(LC-MS/MS) technique to analyze the potential biomarkers in the plasma of mice with heart failure with preserved ejection fraction(HFpEF) induced by a high-fat diet(HFD) and nitric oxide synthase inhibitor(Nω-nitro-L-arginine methyl ester hydrochloride, L-NAME) and explore the pharmacological effects and mechanism of Jiming Powder in improving HFpEF. Male C57BL/6N mice aged eight weeks were randomly assigned to a control group, a model group, an empagliflozin(10 mg·kg~(-1)·d~(-1)) group, and high-and low-dose Jiming Powder(14.3 and 7.15 g·kg~(-1)·d~(-1)) groups. Mice in the control group were fed on a low-fat diet, and mice in the model group and groups with drug intervention were fed on a high-fat diet. All mice had free access to water, with water in the model group and Jiming Powder groups being supplemented with L-NAME(0.5 g·L~(-1)). Drugs were administered on the first day of modeling, and 15 weeks later, blood pressure and cardiac function of the mice in each group were measured. Heart tissues were collected for hematoxylin-eosin(HE) staining to observe pathological changes and Masson's staining to observe myocardial collagen deposition. Untargeted metabolomics analysis was performed on the plasma collected from mice in each group, and metabolic pathway analysis was conducted using MetaboAnalyst 5.0. The results showed that the blood pressure was significantly lower and the myocardial concentric hypertrophy and left ventricular diastolic dysfunction were significantly improved in both the high-dose and low-dose Jiming Powder groups as compared with those in the model group. HE and Masson staining showed that both high-dose and low-dose Jiming Powder significantly alleviated myocardial fibrosis. In the metabolomics experiment, 23 potential biomarkers were identified and eight strongly correlated metabolic pathways were enriched, including linoleic acid metabolism, histidine metabolism, alpha-linolenic acid metabolism, glycerophospholipid metabolism, purine metabolism, porphyrin and chlorophyll metabolism, arachidonic acid metabolism, and pyrimidine metabolism. The study confirmed the pharmacological effects of Jiming Powder in lowering blood pressure and ameliorating HFpEF and revealed the mechanism of Jiming Powder using the metabolomics technique, providing experimental evidence for the clinical application of Jiming Powder in treating HFpEF and a new perspective for advancing and developing TCM therapy for HFpEF.
Male ; Mice ; Animals ; Heart Failure/metabolism* ; Powders ; Stroke Volume/physiology* ; Chromatography, Liquid ; NG-Nitroarginine Methyl Ester/therapeutic use* ; Mice, Inbred C57BL ; Tandem Mass Spectrometry ; Metabolomics ; Biomarkers ; Water

Chronic heart failure is the end stage of heart diseases caused by multiple causes. Myocardial cell injury is the key cause of cardiac function deterioration. Ferroptosis, an iron-dependent programmed death mode, is characterized by iron overload and excessive accumulation of lipid peroxides. Studies have demonstrated that inhibiting ferroptosis has a protective effect on myocardial cells. The theory of "harmful hyperactivity and responding inhibition" is an important rule developed by physicians to explain the generation and restriction of the five elements and the pathological imbalance of the human body, and can guide medication. Correlating with the nature, humans need to rely on the law of responding inhibition to maintain the harmony of five Zang-organs and the steady state of Fu-organs. The pathogenesis of ferroptosis in chronic heart failure highly coincides with the process of failing to "inhibition and hyperactivity becoming harmful". The initial factor of ferroptosis is the deficiency of heart Qi, which results in the inability to maintain the balance of cardiomyocyte redox system. The involvement of the five Zang-organs leads to the loss of distribution of body fluid and blood. As a result, the phlegm turbidity, blood stasis, and water retention in the meridians occur, which are manifested as the accumulation of iron and lipid peroxides, which is the aggravating factor of ferroptosis. The two factors interact with each other, leading to the spiral development and thus aggravating heart failure. According to the traditional Chinese medicine(TCM) pathogenesis of ferroptosis, the authors try to treat the chronic heart failure by stages in accordance with the general principle of restraining excess and alleviating hyperactivity. The early-stage treatment should "nourish heart Qi, regulate the five Zang-organs, so as to restrain excess". The middle-stage treatment should "active blood, resolve phlegm, dispel pathogen, and eliminate turbidity", so as to alleviate hyperactivity. The late-stage treatment should "warm Yang, replenish Qi, active blood, and excrete water". Following the characteristics of pathogenesis, the TCM intervention can reduce iron accumulation and promote the clearance of lipid peroxide, thus inhibiting ferroptosis and improving cardiac function.
Humans ; Ferroptosis ; Lipid Peroxides ; Medicine, Chinese Traditional ; Heart Failure/drug therapy* ; Chronic Disease ; Iron ; Water

This study aims to investigate the hepatotoxicity of Psoraleae Fructus water extract and the underlying mechanism in rats. Forty-eight rats were randomly assigned into four groups: a blank group and low-(BZGL, 6.25 g·kg~(-1)), medium-(BGZM, 12.5 g·kg~(-1)), and high-dose(BGZH, 25 g·kg~(-1)) Psoraleae Fructus water extract groups. The rats were treated for 28 days, and toxicity and mortality were observed daily. After 28 days, the rats were sacrificed, and the body weight, liver index, and liver-to-brain ratio were calculated. The morphological changes in the liver tissue were observed, and the serum levels of related biochemical indicators were measured. The results showed that compared with the blank group, Psoraleae Fructus water extracts of different doses decreased the body weight, increased the liver index and liver-to-brain ratio, and caused liver hypertrophy and pathological changes. Pathological examination revealed that the rats in Psoraleae Fructus water extract groups had bile duct hyperplasia, inflammatory cell infiltration, and liver cell fibrosis. Compared with the blank group, BGZL elevated the levels of alanine transaminase(ALT), α-glutathione S-transferase(α-GST), and total bile acid(TBA)(P<0.05), and BGZM and BGZH elevated the levels of ALT, TBA, α-GST, γ-glutamyl transferase(γ-GT), purine nucleoside phosphorylase(PNP), ornithine carbamoyltransferase(OCT), and arginase(ArgI)(P<0.05). Compared with the blank group, Psoraleae Fructus water extracts of different doses down-regulated the mRNA and protein levels of bile salt export pump(BSEP) and farnesoid X receptor(FXR) and up-regulated the mRNA and protein levels of tumor necrosis factor-α(TNF-α), nuclear factor kappaB(NF-κB), and cholesterol 7 alpha-hydroxylase(CYP7A1)(P<0.05). The results suggested that Psoraleae Fructus water extract caused toxicity in rats, showing a dose-toxicity relationship. Psoraleae Fructus water extract may cause liver damage, which may be due to its effect on liver bile acid secretion and induction of inflammation.
Rats ; Animals ; Water ; Rats, Sprague-Dawley ; Liver ; NF-kappa B ; Liver Cirrhosis ; Bile Acids and Salts ; Body Weight ; RNA, Messenger

This study aims to investigate the mechanism of Linderae Radix water extract(LRWE) in the prevention and treatment of diarrhea-predominant irritable bowel syndrome(IBS-D) based on serum metabolomics. Eighteen 2-week-old male SD rats were randomized into control, IBS-D model, and LRWE groups. The rats in other groups except the control group received gavage of senna concentrate combined with restraint stress for the modeling of IBS-D. The rats in the LRWE group were administrated with LRWE(5.4 g·kg~(-1)) by gavage, and those in the control and IBS-D model groups with an equal volume of distilled water for a total of 14 days. The visceral sensitivity was evaluated by the abdominal withdrawal reflex(AWR) score, and the degree of diarrhea was assessed by the fecal water content(FWC). The morphological changes of the colon and the morphology and number of goblet cells were observed by hematoxylin-eosin(HE) and periodic acid-schiff(PAS) staining, respectively. Ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was used for the screening of the potential biomarkers in the rat serum and their related metabolic pathways. The results showed that LRWE reduced the AWR score, decreased FWC, and alleviated visceral sensitivity and diarrhea symptoms in IBS-D rats. HE and PAS staining showed that LRWE mitigated low-grade intestinal inflammation and increased the number of mature secretory goblet cells in the colonic epithelium of IBS-D rats. A total of 25 potential biomarkers of LRWE in treating IBS-D were screened out in this study, which were mainly involved in riboflavin, tryptophan, glycine, serine and threonine metabolism, glyoxylate and dicarboxylate metabolism, and cysteine and methionine metabolism. The regulatory effects were the most significant on the riboflavin and tryptophan metabolism pathways. LRWE may alleviate the visceral hypersensitivity by promoting energy metabolism and amino acid metabolism, enhancing intestinal barrier function, and improving intestinal immune function in IBS-D rats.
Rats ; Male ; Animals ; Irritable Bowel Syndrome/metabolism* ; Water ; Chromatography, Liquid ; Tryptophan ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; Diarrhea/drug therapy* ; Biomarkers ; Riboflavin