This study aims to explore the role and mechanism of berberine in promoting the expression of aquaporin 4(AQP4) in astrocytes by regulating the expression of miR-383-5p in HepG2 cell-derived exosomes under insulin resistance(IR). The IR-HepG2 cell model was established with 1×10~(-6) mol·L~(-1) insulin. With metformin as the positive control, the safe concentrations of berberine and metformin were screened by cell counting kit-8(CCK-8) and lactate dehydrogenase(LDH) leakage assays, and the effect of berberine on the IR of HepG2 cells was evaluated by glucose consumption. NanoSight was used to measure the particle size and concentration of exosomes secreted by HepG2 cells in each group. HepG2 cell-derived exosomes in each group were incubated with astrocytes for 24 h, and the protein and mRNA levels of AQP4 in HA1800 cells were determined by Western blot and qRT-PCR, respectively. qRT-PCR was performed to determine the expression of miR-383-5p in HepG2 cell-derived exosomes and HA1800 cells after co-incubation. Western blotting was employed to determine the expression levels of miRNAs and proteins associated with exosome production and release in HepG2 cells. The results showed that 10 μmol·L~(-1) berberine and 1 mmol·L~(-1) metformin significantly alleviated the IR of HepG2 cells and reduced the concentration of exosomes in HepG2 cells. The exosomes of HepG2 cells treated with berberine and metformin significantly up-regulated the protein and mRNA levels of AQP4 in HA1800 cells. The mRNA level of miR-383-5p in HepG2 cell exosomes and HA1800 cells co-incubated with berberine and metformin decreased significantly. The intervention with berberine and metformin significantly down-regulated the expression of proteins associated with the production of miRNAs(Dicer, Drosha) as well as the production(Alix, Vps4A) and release(Rab35, VAMP3) of exosomes in IR-HepG2 cells. In conclusion, berberine can promote the expression of AQP4 in astrocytes by inhibiting the production and release of miR-383-5p in HepG2-derived exosomes under IR.
Humans ; MicroRNAs/metabolism* ; Berberine/pharmacology* ; Hep G2 Cells ; Exosomes/genetics* ; Aquaporin 4/metabolism* ; Insulin Resistance ; Astrocytes/drug effects*

This paper aims to investigate the hypoglycemic effect and mechanism of berberine in improving energy metabolism based on the multi-pathway regulation of brain and muscle aromatic hydrocarbon receptor nuclear translocal protein 1(BMAL1): cyclin kaput complex of day-night spontaneous output cyclin kaput(CLOCK). The dexamethasone-induced hepatic insulin resistance(IR) HepG2 cell model was used; 0.5, 1, 5, 10, 20 μmol·L~(-1) berberine were administered at 15, 18, 21, 24, 30, 36 h. The time-dose effect of glucose content in extracellular fluid was detected by glucose oxidase method. The optimal dosage and time of berberine were determined for the follow-up study. Glucose oxidase method and chemiluminescence method were respectively performed to detect hepatic glucose output and relative content of ATP in cells; Ca~(2+), reactive oxygen species(ROS), mitochondrial structure and membrane potential were detected by fluorescent probes. Moreover, ultraviolet colorimetry method was used to detect the liver type of pyruvate kinase(L-PK) and phosphoenol pyruvate carboxykinase(PEPCK). In addition, pyruvate dehydrogenase E1 subunit α1(PDHA1), phosphate fructocrine-liver type(PFKL), forkhead box protein O1(FoxO1), peroxisome proliferator-activated receptor gamma co-activator 1α(PGC1α), glucose-6-phosphatase(G6Pase), glucagon, phosphorylated nuclear factor-red blood cell 2-related factor 2(p-Nrf2)(Ser40), heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), fibroblast growth factor 21(FGF21), uncoupled protein(UCP) 1 and UCP2 were detected by Western blot. BMAL1:CLOCK complex was detected by immunofluorescence double-staining method, combined with small molecule inhibitor CLK8. Western blot was used to detect PDHA1, PFKL, FoxO1, PGC1α, G6Pase, glucagon, Nrf2, HO-1, NQO1, FGF21, UCP1 and UCP2 in the CLK8 group. The results showed that berberine downregulated the glucose content in extracellular fluid in IR-HepG2 cells in a time-and dose-dependent manner. Moreover, berberine inhibited hepatic glucose output and reduced intracellular Ca~(2+) and ROS whereas elevated JC-1 membrane potential and improved mitochondrial structure to enhance ATP production. In addition, berberine upregulated the rate-limiting enzymes such as PFKL, L-PK and PDHA1 to promote glycolysis and aerobic oxidation but also downregulated PGC1α, FoxO1, G6Pase, PEPCK and glucagon to inhibit hepatic gluconeogenesis. Berberine not only upregulated p-Nrf2(Ser40), HO-1 and NQO1 to enhance antioxidant capacity but also upregulated FGF21, UCP1 and UCP2 to promote energy metabolism. Moreover, berberine increased BMAL1, CLOCK and nuclear BMAL1:CLOCK complex whereas CLK8 reduced the nuclear BMAL1:CLOCK complex. Finally, CLK8 decreased PDHA1, PFKL, Nrf2, HO-1, NQO1, FGF21, UCP1, UCP2 and increased FoxO1, PGC1α, G6Pase and glucagon compared with the 20 μmol·L~(-1) berberine group. BMAL1:CLOCK complex inhibited gluconeogenesis, promoted glycolysis and glucose aerobic oxidation pathways, improved the reduction status within mitochondria, protected mitochondrial structure and function, increased ATP energy storage and promoted energy consumption in IR-HepG2 cells. These results suggested that berberine mediated BMAL1:CLOCK complex to coordinate the regulation of hepatic IR cells to improve energy metabolism in vitro.
Humans ; Berberine/pharmacology* ; Gluconeogenesis/drug effects* ; Hep G2 Cells ; Glucose/metabolism* ; Liver/drug effects* ; Energy Metabolism/drug effects* ; Hypoglycemic Agents/pharmacology* ; ARNTL Transcription Factors/genetics* ; Glycolysis/drug effects* ; Oxidation-Reduction/drug effects*

Objective This study aims to investigate the effect of berberine (Ber) on foam cell formation induced by oxidized low-density lipoprotein (ox-LDL) in macrophages and to explore the mechanism's association with the ACE2-Ang(1-7)-Mas axis. Methods They were randomly divided into blank group, model group (RAW264.7 cells induced with 60 μg/mL ox-LDL), and berberine group (the model treated with berberine interventions at 2.5, 5, and 10 μmol/L concentrations). Lipid accumulation within the cells was assessed by Oil Red O staining, and the content of lipid droplets in each group was quantitatively analyzed by enzymatic method. The content of total cholesterol (TC) and free cholesterol (FC) in foam cells were detected by enzymatic method. The levels of oxidative stress factors (malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH)), inflammatory factors such as tumor necrosis factor α(TNF-α), and nitric oxide (NO) were measured using corresponding relevant reagent kits. The mRNA and protein expressions of ACE2 and Mas were evaluated through quantitative real-time PCR and Western blot analysis, respectively. The levels of AngII and Ang(1-7) were detected by ELISA. Results Compared with the model group, the berberine groups exhibited reduced lipid droplet accumulation and a dose-dependent decrease in intracellular lipid content. Berberine significantly lowered TC and FC levels in foam cells and reduced the CE/TC ratio. The levels of the oxidative factor MDA were significantly reduced, while the levels of the antioxidant factors SOD and GSH were markedly increased. Inflammatory factors TNF-α and NO were significantly decreased. The expression of the ACE2-Ang(1-7)-Mas signaling pathway was significantly activated, and the effect was more pronounced in the Ber group with high-concentration compared to the group with low-concentration, demonstrating a dose-dependent response. Conclusion Berberine can inhibit macrophage foam cell formation, potentially through upregulation of the ACE2-Ang(1-7)-Mas signaling pathway, thereby contributing to the alleviation of atherosclerosis.
Berberine/pharmacology* ; Foam Cells/cytology* ; Animals ; Signal Transduction/drug effects* ; Mice ; Angiotensin-Converting Enzyme 2 ; Angiotensin I/genetics* ; Peptidyl-Dipeptidase A/genetics* ; Peptide Fragments/genetics* ; Receptors, G-Protein-Coupled/genetics* ; RAW 264.7 Cells ; Proto-Oncogene Proteins/genetics* ; Proto-Oncogene Mas ; Lipoproteins, LDL/pharmacology* ; Nitric Oxide/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVE: To investigate the hepatotoxicity of alkaloids from Euodiae Fructus combined with berberine (BBR) in Zuojin Pill, and to preliminarily explore the possible detoxification mechanism of the combination components. METHODS: The combination ratio of components was determined by the maximum concentration (Cmax) of the chemical components in Zuojin Pill. HepG2 cell model was used to investigate the combined toxicity of the hepatotoxic components from Euodiae Fructus, such as evodiamine (EVO) or dehydroevodiamine (DHED), with BBR for 48 h. The experimental groups were set as follows: the vehicle control group, the EVO group, the DHED group, the BBR group, and the combination group of EVO or DHED with BBR. The cell counting kit-8 (CCK-8) method was used to determine the cell viability, and the combination index (CI) was used to determine the combined toxicity of the components. The alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydroge-nase (LDH), and alkaline phosphatase (ALP) activities as well as total bilirubin (TBIL) content in the cell culture supernatant were detected. The protein expression levels of bile acid transporters, such as bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2), were detected by Western blot. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in HepG2 cells were detected. RESULTS: Compared with EVO or DHED group, the combination of EVO 1 μmol/L with BBR 10 μmol/L or DHED 50 μmol/L with BBR 35 μmol/L significantly increased cell viability of HepG2 cells (P < 0.01), with CI values of 77.89 or 4.49, respectively, much greater than 1. Significant decreases in the activities of ALT, AST, LDH, ALP, and TBIL content in the cell culture supernatant were found in both combination groups (P < 0.05, P < 0.01). Compared with the EVO group, the combination of EVO with BBR upregulated the protein expression levels of BSEP and MRP2. Compared with the DHED group, the combination of DHED with BBR significantly downregulated the protein expression levels of BSEP and MRP2 (P < 0.01). Compared with EVO or DHED group, the combination of EVO or DHED with BBR significantly reduced the MDA content in HepG2 cells (P < 0.05, P < 0.01). CONCLUSION A certain ratio of BBR combined with EVO or DHED had an antagonistic effect on HepG2 cytotoxicity, which might be related to regulating the expression of bile acid transpor-ters, and reducing lipid peroxidation damage.
Humans ; Hep G2 Cells ; Berberine/pharmacology* ; Drugs, Chinese Herbal/toxicity* ; Evodia/chemistry* ; Alkaloids/pharmacology* ; Cell Survival/drug effects* ; Multidrug Resistance-Associated Proteins/metabolism* ; Multidrug Resistance-Associated Protein 2 ; Quinazolines

OBJECTIVES: To determine the neuroprotective effects of berberine hydrochloride (BBR) against lead-induced injuries on the hippocampus of rats. METHODS: Wistar rats were exposed orally to doses of 100 and 500 ppm lead acetate for 1 and 2 months to develop subchronic and chronic lead poisening models, respectively. For treatment, BBR (50 mg/kg daily) was injected intraperitoneally to rats poisoned with lead. At the end of the experiment, the spatial learning and memory of rats were assessed using the Morris water maze test. Hippocampal tissue changes were examined by hematoxylin and eosin staining. The activity of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase, and malondialdehyde levels as parameters of oxidative stress and antioxidant status of the hippocampus were evaluated. RESULTS: BBR reduced cognitive impairment in rats exposed to lead (P<0.05 or P<0.01). The resulting biochemical changes included a decrease in the activity of antioxidants and an increase in lipid peroxidation of the hippocampus of lead-exposed rats (P<0.05 or P<0.01), which were significantly modified by BBR (P<0.05). BBR also increased the density of healthy cells in the hippocampus of leadexposed rats (P<0.05). Significant changes in tissue morphology and biochemical factors of the hippocampus were observed in rats that received lead for 2 months (P<0.05). Most of these changes were insignificant in rats that received lead for 1 month. CONCLUSION BBR can improve oxidative tissue changes and hippocampal dysfunction in lead-exposed rats, which may be due to the strong antioxidant potential of BBR.
Animals ; Hippocampus/pathology* ; Rats, Wistar ; Antioxidants/pharmacology* ; Berberine/therapeutic use* ; Cognition/drug effects* ; Male ; Lead Poisoning/metabolism* ; Chronic Disease ; Oxidative Stress/drug effects* ; Maze Learning/drug effects* ; Rats ; Lipid Peroxidation/drug effects* ; Malondialdehyde/metabolism*

This study aims to investigate the mechanism of berberine in regulating the metabolism network via clock-controlled genes represented by brain and muscle arnt-like 1(BMAL1) to ameliorate insulin resistance(IR) of hepatocytes in vitro. The HepG2 cell model of dexamethasone-induced IR(IR-HepG2) was established and treated with 5, 10, and 20 μmol·L~(-1) berberine, respectively, for 24 h. The glucose oxidase method and cell counting kit-8(CCK-8) assay were employed to measure extracellular glucose concentration and cell viability, respectively. Periodic acid-Schiff(PAS) staining and lipid fluorescence method were used to detect glycogen and lipids. The immunofluorescence(IF) assay was employed to detect the nuclear localization of BMAL1 and circadian locomotor output cycles kaput(CLOCK) in IR-HepG2 cells. Western blot was employed to determine the protein levels of BMAL1, CLOCK, period circadian clock 2(PER2), cryptochrome circadian regulator 1(CRY1), Rev-Erbα, carbohydrate response element-binding protein(ChREBP), peroxisome proliferator-activated receptors alpha and gamma(PPARα/γ), sterol regulatory element-binding protein 1C(SREBP-1C), mammalian target of rapamycin(mTOR), protein kinase B(Akt), glycogen synthase kinase-3β(GSK3β), acetyl coenzyme A carboxylase 1(ACC1), fatty acid synthase(FASN), carnitine palmitoyltransferase 1α(CPT1α), nicotinamide phosphoribosyltransferase(NAMPT), silent information regulator 1(SIRT1), adiponectin(ADPN), insulin receptor substrate 2(IRS2), and phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85). In addition, the levels of phosphorylated adenosine monophosphate-activated protein kinase alpha(AMPKα), Akt, GSK3β, BMAL1, and mTOR were determined. Furthermore, 20 μmol·L~(-1) CLK8 was added to measure the glucose consumption as well as the protein levels of ChREBP, PPARα, and mTOR in IR-HepG2 cells. The results showed that berberine increased the glucose consumption, lowered the lipid levels, increased the expression and nuclear localization of BMAL1 and CLOCK, and up-regulated the level of BMAL1 in IR-HepG2 cells. Furthermore, berberine up-regulated the levels of ADPN, IRS2, PI3Kp85, p-Akt(Ser473)/Akt, p-mTOR(Ser2448)/mTOR, PPARα, and CPT1α, and down-regulated the levels of p-GSK3β(Ser9)/GSK3β, ChREBP, SREBP-1C, ACC1, and FASN. The addition of CLK8 reduced glucose consumption in IR-HepG2 cells, up-regulated the ChREBP level, and down-regulated PPARα and mTOR levels by inhibiting the BMAL1 and CLOCK interaction. In summary, berberine regulated glucose and lipid metabolism via clock-controlled genes with BMAL1 at the core to ameliorate IR of hepatocytes.
Humans ; Hepatocytes/drug effects* ; Lipid Metabolism/drug effects* ; Glucose/metabolism* ; Berberine/pharmacology* ; Insulin Resistance ; Hep G2 Cells ; CLOCK Proteins/genetics* ; ARNTL Transcription Factors/genetics*

The Compound Cheqian Tablets are derived from Cheqian Power in Comprehensive Recording of Divine Assistance, and they are made by modern technology with the combination of Plantago asiatica and Coptis chinensis. To investigate the material basis of Compound Cheqian Tablets in the treatment of diabetic nephropathy, in this study, the chemical components of Compound Cheqian Tablets were characterized and analyzed by UPLC-Q-TOF-MS/MS, and a total of 48 chemical components were identified. The identified chemical compounds were analyzed by network pharmacology. By validating with previous literature, six bioactive compounds including acteoside, isoacteoside, coptisine, magnoflorine, palmatine, and berberine were confirmed as the index components for qua-lity evaluation. Furthermore, the content of the six components in the Compound Cheqian Tablets was determined by the "double external standards" quantitative analysis of multi-components by single marker(QAMS), and the relative correction factor of isoacteoside was calculated as 1.118 by using acteoside as the control; the relative correction factors of magnoflorine, palmatine, and berberine were calculated as 0.729, 1.065, and 1.126, respectively, by using coptisine as the control, indicating that the established method had excellent stability under different conditions. The results obtained by the "double external standards" QAMS approximated those obtained by the external standard method. This study qualitatively characterized the chemical components in the Compound Cheqian Tablets by applying UPLC-Q-TOF-MS/MS and screened the pharmacodynamic substance basis for the treatment of diabetic nephropathy via network pharmacology, and primary pharmacodynamic substance groups were quantitatively analyzed by the "double external stan-dards" QAMS method, which provided a scientific basis for clarifying the pharmacodynamic substance basis and quality control of Compound Cheqian Tablets.
Humans ; Tandem Mass Spectrometry ; Berberine/pharmacology* ; Chromatography, High Pressure Liquid/methods* ; Network Pharmacology ; Diabetic Nephropathies ; Drugs, Chinese Herbal/chemistry* ; Quality Control ; Tablets

Scutellariae Radix-Coptidis Rhizoma(SR-CR) herbal pair is commonly used in many compound prescriptions for their synergistic heat-clearing and dampness-drying properties. During the decoction process, a substantial amount of precipitate is generated. However, there have been no explicit reports on the composition, morphology, and potential effects of this precipitate on the in vivo behavior of SR-CR decoction. This study employed high-performance liquid chromatography(HPLC), high-resolution mass spectrometry, and other techniques to analyze the composition of the co-precipitate in the decoction of SR-CR. Scanning electron microscopy and mass spectrometry imaging were used to analyze its appearance and morphology. Additionally, rats were used to investigate the effects of the co-precipitate on the in vivo behavior of the main components in the SR-CR decoction. The research findings indicated that eight components, including coptisine, berberine, epiberberine, palmatine, baicalin, oroxylin A-7-O-β-D-glucuronide, wogonoside and baicalein, constituted the primary composition of the co-precipitate. Among these, baicalin and berberine hydrochloride were the most abundant, accounting for about 60% of the total weight. Moreover, the co-precipitate contained 18% tannins. Morphological analysis revealed that the particles in the SR-CR decoction precipitate were spherical microparticles with an average diameter of around 600 nm. Pharmacokinetic research demonstrated that there were significant differences in the AUC, C_(max), t_(1/2), and T_(max) of baicalin, a major component, in rats administered with lyophilized powders of the combined decoction and single decoctions of SR-CR orally, suggesting that the precipitate generated during the decoction process can affect the in vivo behavior of the main components of the SR-CR decoction. It can reduce the absorption of baicalin in the body, decrease the extent of rapid drug release, and to a certain extent, prevent adverse reactions or side effects.
Rats ; Animals ; Drugs, Chinese Herbal/pharmacology* ; Scutellaria baicalensis/chemistry* ; Berberine ; Chromatography, High Pressure Liquid ; Mass Spectrometry

Ulcerative colitis(UC) is a recurrent, intractable inflammatory bowel disease. Coptidis Rhizoma and Bovis Calculus, serving as heat-clearing and toxin-removing drugs, have long been used in the treatment of UC. Berberine(BBR) and ursodeoxycholic acid(UDCA), the main active components of Coptidis Rhizoma and Bovis Calculus, respectively, were employed to obtain UDCA-BBR supramolecular nanoparticles by stimulated co-decocting process for enhancing the therapeutic effect on UC. As revealed by the characterization of supramolecular nanoparticles by field emission scanning electron microscopy(FE-SEM) and dynamic light scattering(DLS), the supramolecular nanoparticles were tetrahedral nanoparticles with an average particle size of 180 nm. The molecular structure was described by ultraviolet spectroscopy, fluorescence spectroscopy, infrared spectroscopy, high-resolution mass spectrometry, and hydrogen-nuclear magnetic resonance(H-NMR) spectroscopy. The results showed that the formation of the supramolecular nano-particle was attributed to the mutual electrostatic attraction and hydrophobic interaction between BBR and UDCA. Additionally, supramolecular nanoparticles were also characterized by sustained release and pH sensitivity. The acute UC model was induced by dextran sulfate sodium(DSS) in mice. It was found that supramolecular nanoparticles could effectively improve body mass reduction and colon shortening in mice with UC(P<0.001) and decrease disease activity index(DAI)(P<0.01). There were statistically significant differences between the supramolecular nanoparticles group and the mechanical mixture group(P<0.001, P<0.05). Enzyme-linked immunosorbent assay(ELISA) was used to detect the serum levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6), and the results showed that supramolecular nanoparticles could reduce serum TNF-α and IL-6 levels(P<0.001) and exhibited an obvious difference with the mechanical mixture group(P<0.01, P<0.05). Flow cytometry indicated that supramolecular nanoparticles could reduce the recruitment of neutrophils in the lamina propria of the colon(P<0.05), which was significantly different from the mechanical mixture group(P<0.05). These findings suggested that as compared with the mechanical mixture, the supramolecular nanoparticles could effectively improve the symptoms of acute UC in mice. The study provides a new research idea for the poor absorption of small molecules and the unsatisfactory therapeutic effect of traditional Chinese medicine and lays a foundation for the research on the nano-drug delivery system of traditional Chinese medicine.
Animals ; Mice ; Colitis, Ulcerative/drug therapy* ; Ursodeoxycholic Acid/adverse effects* ; Berberine/pharmacology* ; Interleukin-6 ; Tumor Necrosis Factor-alpha/pharmacology* ; Drugs, Chinese Herbal/pharmacology* ; Colon ; Nanoparticles ; Dextran Sulfate/adverse effects* ; Disease Models, Animal ; Colitis/chemically induced*

Objective: To explore the impact of traditional Chinese medicine berberine (BBR) on membrane integrity and permeability of Methicillin-resistant Staphylococcus aureus (MRSA) and the change of bacterial cell wall structure, laying a foundation for the clinical application of berberine in antibacterial. Methods: This study used a non-randomized concurrent controlled trial. The 3 MRSA strains were isolated and cultured from lower respiratory tract samples of geriatric patients from Shanghai Eighth People's Hospital between 2019 and 2020.The Meirier VETEK MS fully automated rapid microbial mass spectrometry detection system and VETEK 2 Compact fully automated microbial identification instrument were used to identify bacterial drug sensitivity experiments to detect bacterial species and drug sensitivity. The minimal inhibitory concentration (MIC) of BBR on MRSA strains was determined by broth microdilution. This study used conductivity tests to assess the changes in membrane permeability in response to different concentration of BBR on MRSA, while also investigating the changes in MRSA morphology by transmission electron microscopy. GraphPad Prism5 was used to analyze the differences in the electrical conductivity experimental results. Results: The MIC of BBR on MRSA was 64 μg/ml. After co-culturing MRSA with BBR for 4 h at 8 μg/ml, 16 μg/ml, 32 μg/ml, 64 μg/ml and 128 μg/ml, respectively, the electrical conductivity increased, compared with the control group, by 24.49%,34.59%,208.92%,196.40% and 208.68%, respectively. By transmission electron microscopy, This study found that low concentration of BBR (8 μg/ml,1/8 MIC) caused no significant damage to MRSA, and the bacterial structure of MRSA remained intact. The cell wall of MRSA became thinner after treatment with berberine at medium concentration (64 μg/ml,1 MIC), while high concentration of BBR (512 μg/ml,8 MIC) induced the destruction and dissolution of MRSA cell wall structure and the leakage of bacterial contents, leading to bacterial lysis. Conclusion: Berberine can kill bacteria by altering the permeability of MRSA cell membrane and destroying and dissolving the structure of the cell wall.
Methicillin-Resistant Staphylococcus aureus ; Berberine/pharmacology* ; China ; Anti-Bacterial Agents/pharmacology* ; Cell Membrane ; Microbial Sensitivity Tests