Combined Use of Astragalus Polysaccharide and Berberine Attenuates Insulin Resistance in IR-HepG2 Cells via Regulation of the Gluconeogenesis Signaling Pathway.
ABSTRACT: Insulin resistance (IR) is likely to induce metabolic syndrome and type 2 diabetes mellitus (T2DM). Gluconeogenesis (GNG) is a complex metabolic process that may result in glucose generation from certain non-carbohydrate substrates. Chinese herbal medicine astragalus polysaccharides and berberine have been documented to ameliorate IR, and combined use of astragalus polysaccharide (AP) and berberine (BBR) are reported to synergistically produce an even better effect. However, what change may occur in the GNG signaling pathway of IR-HepG2 cells in this synergistic effect and whether AP-BBR attenuates IR by regulating the GNG signaling pathway remain unclear. For the first time, we discovered in this study that the optimal time of IR-HepG2 cell model formation was 48 h after insulin intervention. AP-BBR attenuated IR in HepG2 cells and the optimal concentration was 10 mg. AP-BBR reduced the intracellular H2O2 content with no significant effect on apoptosis of IR-HepG2 cells. In addition, a rapid change was observed in intracellular calcium current of the IR-HepG2 cell model, and AP-BBR intervention attenuated this change markedly. The gene sequencing results showed that the GNG signaling pathway was one of the signaling pathways of AP-BBR to attenuate IR in IR-Hepg2 cells. The expression of p-FoxO1Ser256 and PEPCK protein was increased, and the expression of GLUT2 protein was decreased significantly in the IR-HepG2 cell model, and both of these effects could be reversed by AP-BBR intervention. AP-BBR attenuated IR in IR-HepG2 cells, probably by regulating the GNG signaling Pathway.
Project description:Insulin resistance (IR) is likely to induce metabolic syndrome and type 2 diabetes mellitus (T2DM). Gluconeogenesis (GNG) is a complex metabolic process that may result in glucose generation from certain non-carbohydrate substrates. Chinese herbal medicine astragalus polysaccharides and berberine have been documented to ameliorate IR, and combined use of astragalus polysaccharide (AP) and berberine (BBR) are reported to synergistically produce an even better effect. However, what change may occur in the GNG signaling pathway of IR-HepG2 cells in this synergistic effect and whether AP-BBR attenuates IR by regulating the GNG signaling pathway remain unclear. For the first time, we discovered in this study that the optimal time of IR-HepG2 cell model formation was 48 hours after insulin intervention. AP-BBR attenuated IR in HepG2 cells and the optimal concentration was 10mg. AP-BBR reduced the intracellular H2O2 content with no significant effect on apoptosis of IR-HepG2 cells. In addition, a rapid change was observed in intracellular calcium current of the IR-HepG2 cell model, and AP-BBR intervention attenuated this change markedly. The gene sequencing results showed that the GNG signaling pathway was one of the signaling pathways of AP-BBR to attenuate IR in IR-Hepg2 cells. The expression of p-FoxO1Ser256 and PEPCK protein was increased and the expression of GLUT2 protein was decreased significantly in the IR-HepG2 cell model, and both of these effects could be reversed by AP-BBR intervention. AP-BBR attenuated IR in IR-HepG2 cells, probably by regulating the GNG signaling Pathway. Overall design: HepG2 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in 1640 medium (Hyclone, Beijing, China) containing 10% fetal bovine serum (FBS, Hyclone, Beijing, China) and 1 × streptomycin in a 37 ℃ 5% CO2 saturated humidity incubator. The normally cultured HepG2 cell lines in log phase were centrifuged at 100grpm for 5 min, and 20000 cells/well were placed in a 96-well plate and incubated at 37°C.The experiment was performed in 24-h control group, 24-h model group, 36-h control group, 36-h model group, 48-h control group, 48-h model group, 72-h control group and 72-h model group. Insulin (Gibco, NY, USA) was diluted to a final concentration of 10-6 mol/L in complete medium. 200µl insulin preparation was added into each well for the model group and an equal amount of complete medium was added into each well for the control group. Culture was performed in a 37 ℃ 5% CO2 and saturated humidity incubator. The supernatant of the corresponding medium was collected according to the time point by centrifugation at 3000 r/min for 5 min and stored at-80 ℃ for use. RNA isolation, purification and quantification: Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Agilent 2100 with RIN number >7.0.cDNA Library Construction: Poly(A) RNA was purified from total RNA (5ug) using poly-T oligo-attached magnetic beads using two rounds of purification. Then, the poly(A) RNA was fragmented into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA, which was subsequently used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP. An A-base was then added to the blunt ends of each strand, which were prepared for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, the ligated products were amplified by PCR under the following conditions: initial denaturation at 95℃ for 3 min, 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, extension at 72℃ for 30 sec, and then final extension at 72℃ for 5 min. The mean insert size for the final cDNA library was 300 bp (±50 bp). Finally, 150bp paired-end sequencing was performed on an Illumina Hiseq 4000 (LC Bio, China) following the vendor's recommended protocol.Pathway enrichment analysis: Using the DAVID database and mouse genome as background control, the differentially expressed genes were analyzed by gene ontology under "FunctionalAnnotation Chart" functional module. The differentially expressed genes were divided into three categories according to their functions: the biological process, the cell component and the molecular function. Pathway analysis was carried out by KEGG analysis function.
Project description:Background:Radix Astragali (Astragalus membranaceus var. mongholicus (Bunge)) and Coptis chinensis (Coptis chinensis var. angustiloba) are two commonly prescribed traditional Chinese herbs for diabetes. Astragalus Polysaccharide (AP) and Berberine (BBR) are active ingredients of these two herbs respectively and they are scientifically proved to have immunomodulatory and anti-inflammatory effects. They are also known for their antidiabetic potential by ameliorating insulin resistance (IR). AP and BBR have shown different advantages in treating diabetes according to previous reports. However, very few studies focus on the combined activities of the two potential antidiabetic ingredients. In this study, we discovered that reactive oxygen species (ROS) accumulated in IR-hepG2 cells and APBBR can decrease ROS level in model group significantly. We conjectured that APBBR can ameliorate IR in hepG2 cells by decreasing ROS level. In order to verify this hypothesis, we obtained phenotype and transcriptome information of IR-HepG2 cells and explore the underlying mechanism of the combination of AP and BBR(APBBR) activity on the relationship between ROS change in IR at whole-transcriptome level, so as to shed new light to efficacy and application of APBBR in treating diabetes. Methods:The IR cell model was established with high-level insulin intervention. Glucose content, HepG2 cell viability as well as ROS level was detected to study the effect of IR-hepG2 cell phenotype. Unbiased genome-wide RNA sequencing was used to investigate alterations in experimental groups. Then, GO and KEGG functional enrichment was performed to explore the function and pathway of target genes. Venn analysis found out the differentially expressed lncRNAs that had close relationship with IR and ROS. Finally, we screened out candidate lncRNAs and these target genes to construct interaction network of differentiated lncRNA-miRNA-mRNA by according to the principle of competitive endogenous RNA (ceRNA). Results:The biochemical experiments showed that APBBR administration could improve the proliferation activity of IR-HepG2 cells and decrease ROS level in model cells. The GO and KEGG functional enrichment analyses demonstrated several mRNAs remarkably enriched in biological processes and signaling pathways related to ROS production and IR progression. Interaction network suggest that APBBR ameliorates IR in HepG2 cells by regulating the expression of multiple genes and activating relevant signaling pathway to decrease ROS level. Thus, we demonstrated that APBBR ameliorated IR in hepG2 cells via the ROS-dependent pathway.
Project description:Berberine (BBR), a natural compound extracted from a Chinese herb, has been shown to effectively attenuate insulin resistance (IR) and inflammation in the clinic. However, its ameliorative mechanism against IR is not well defined. This study is aimed at investigating the effect of BBR and protein phosphatase, Mg2+/Mn2+-dependent 1B (PPM1B) on IR. Biochemical measurements and liver histopathology were detected using the biochemical analyzer and HE staining in ZDF rats, respectively. Microarray analysis of liver tissues was performed, and differentially expressed gene (DEG) levels were examined by quantitative real-time PCR (qPCR) and Western blot. Additionally, the effect of BBR was also explored in HepG2-IR cells. The glucose oxidase method and the fluorescent glucose analog were used to detect glucose consumption and uptake, respectively. The PKA inhibitor H89, ELISA, qPCR, Western blot, and immunofluorescence staining were employed to estimate the expression levels of related signaling pathways. To evaluate the roles of PPM1B, HepG2-IR cells were stably infected with lentivirus targeting PPM1B. The administration of BBR drastically decreased the body weight, urine volume, blood glucose, blood urea nitrogen (BUN), CHOL, hepatic index levels, and pathologic changes and improved ALB levels in ZDF rats with PPM1B upregulation. Furthermore, BBR effectively improves glucose consumption, uptake, and inflammation in HepG2-IR cells. The knockdown of PPM1B expression aggravated the inflammatory response and glycometabolism disorder in HepG2-IR cells. Mechanistically, a reversal in the expression of cAMP, PKA, PPM1B, PPAR?, LRP1, GLUT4, NF-?B p65, JNK, pIKK? Ser181, IKK?, IRS-1 Ser307, IRS-1, IRS-2 Ser731, IRS-2, PI3K p85, and AKT Ser473 contributes to ameliorate IR in HepG2-IR cells with BBR treatment. Altogether, these results suggest that BBR might regulate IR progression through the regulation of the cAMP, PKA, PPM1B, PPAR?, LRP1, GLUT4, NF-?B p65, JNK, pIKK? Ser181, IKK?, IRS-1 Ser307, IRS-1, IRS-2 Ser731, IRS-2, PI3K p85, and AKT Ser473 expression in the liver.
Project description:Berberine (BBR) is the main component of Coptidis rhizoma, the dried rhizome of Coptis chinensis and is a potential plant alkaloid used for the treatment of cancer due to its high antitumor activity. The present study examined the therapeutic potential and molecular mechanism of action of BBR against HCC, using systematic pharmacology combined with a molecular docking approach and experimental validation in vitro. Through systematic pharmacological analysis, it was found that BBR serves a significant role in inhibiting HCC by affecting multiple pathways, especially the PI3K/AKT signaling pathway. Furthermore, the docking approach indicated that the binding of BBR to AKT could lead to the suppression of AKT activity. The present study examined the inhibitory effect of BBR on the PI3K/AKT pathway in HCC and identified that BBR downregulated the expressions of phosphorylated AKT and PI3K in MHCC97?H and HepG2 cells, inhibiting their growth, cell migration and invasion in a dose?dependent manner. In addition, inhibition of the AKT pathway by BBR also contributed to cell apoptosis in MHCC97?H and HepG2 cells. Taken together, the results of the present study suggested that BBR may be a promising antitumor drug for HCC that acts by inhibiting the PI3K/AKT pathway.
Project description:Berberine (BBR) has been shown to exhibit protective effects against diabetes and dyslipidemia. Previous studies have indicated that BBR modulates lipid metabolism and inhibits hepatic gluconeogensis by decreasing expression of Hepatocyte Nuclear Factor-4α (HNF-4α). However, the mechanism involved in this process was unknown. In the current study, we examined the mechanism of how BBR attenuates hepatic gluconeogenesis and the lipid metabolism alterations observed in type 2 diabetic (T2D) mice and in palmitate (PA)-incubated HepG2 cells. Treatment with BBR for 4 weeks improve all biochemical parameters compared to T2D mice. Treatment of T2D mice for 4 weeks or treatment of PA-incubated HepG2 cells for 24 h with BBR decreased expression of HNF-4α and the microRNA miR122, the key gluconeogenesis enzymes Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase) and the key lipid metabolism proteins Sterol response element binding protein-1 (SREBP-1), Fatty acid synthase-1 (FAS-1) and Acetyl-Coenzyme A carboxylase (ACCα) and increased Carnitine palmitoyltransferase-1(CPT-1) compared to T2D mice or PA-incubated HepG2 cells. Expression of HNF-4α in HepG2 cells increased expression of gluconeogenic and lipid metabolism enzymes and BBR treatment or knock down of miR122 attenuated the effect of HNF-4α expression. In contrast, BBR treatment did not alter expression of gluconeogenic and lipid metabolism enzymes in HepG2 cells with knockdown of HNF-4α. In addition, miR122 mimic increased expression of gluconeogenic and lipid metabolism enzymes in HepG2 cells with knockdown of HNF-4α. These data indicate that miR122 is a critical regulator in the downstream pathway of HNF-4α in the regulation of hepatic gluconeogenesis and lipid metabolism in HepG2 cells. The effect of BBR on hepatic gluconeogenesis and lipid metabolism is mediated through HNF-4α and is regulated downstream of miR122. Our data provide new evidence to support HNF-4α and miR122 regulated hepatic gluconeogenesis and lipid metabolism as promising therapeutic targets for the treatment of T2D.
Project description:<b>Background:</b> Chronic low-grade inflammation is recognized as a key pathophysiological mechanism of insulin resistance. Leukotriene B4 (LTB4), a molecule derived from arachidonic acid, is a potent neutrophil chemoattractant. The excessive amount of LTB4 that is combined with its receptor BLT1 can cause chronic low-grade inflammation, aggravating insulin resistance. Berberine (BBR) has been shown to relieve insulin resistance due to its anti-inflammatory properties. However, it is not clear whether BBR could have any effects on the LTB4-BLT1 axis. <b>Methods:</b> Using LTB4 to induce Raw264.7 and HepG2 cells, we investigated the effect of BBR on the LTB4-BLT1 axis in the progression of inflammation and insulin resistance. <b>Results:</b> Upon exposure to LTB4, intracellular insulin resistance and inflammation increased in HepG2 cells, and chemotaxis and inflammation response increased in RAW264.7 cells. Interestingly, pretreatment with BBR partially blocked these changes. Our preliminary data show that BBR might act on BLT1, modulating the LTB4-BLT1 axis to alleviate insulin resistance and inflammation. <b>Conclusions:</b> Our study demonstrated that BBR treatment could reduce intracellular insulin resistance and inflammation of hepatic cells, as well as chemotaxis of macrophages induced by LTB4. BBR might interact with BLT1 and alter the LTB4-BLT1 signaling pathway. This mechanism might be a novel anti-inflammatory and anti-diabetic function of BBR.
Project description:<h4>Purpose</h4>To evaluate the effect of berberine (BBR) on polycystic ovary syndrome (PCOS) patients with insulin resistance (IR).<h4>Methods</h4>PubMed (in English), Medline (in English), Embase (in English), CNKI (in Chinese), WanFang DATA (in Chinese), and VIP (in Chinese) were searched for randomized controlled trials in human beings with the search terms including "polycystic ovary syndrome /PCOS" and "berberine/BBR/Huang liansu (in Chinese)/ Xiao bojian (in Chinese)" till July 2018. Relevant indices were collected and analyzed by Stata 13.0.<h4>Results</h4>A total of 9 randomized controlled trials were included. Limited data demonstrated the results as follows: No significant difference was found between berberine (BBR) and metformin (MET) on alleviating insulin resistance, improving glycolipid metabolism, or reproductive endocrine condition. MET combined with BBR was not superior to MET alone, but cyproterone acetate (CPA) combined with BBR was superior to CPA alone in improving some of the reproductive endocrine indices. The combination of BBR and Chinese herbs also showed positive effect. However there are insufficient data to make any conclusions on the effect of BBR on PCOS-IR.<h4>Conclusion</h4>BBR showed a promising prospect in treating PCOS-IR. But its mechanisms are still unclear, and more properly designed, randomized, double-blind, placebo-controlled trials are needed to further confirm its effect and safety.
Project description:The aim of the present study was to investigate the effects of advanced glycation end products (AGEs) and berberine hydrochloride (BBR) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs) in vitro, and their underlying mechanisms. hPDLSCs were subjected to osteogenic induction and were treated with AGEs or AGEs + BBR. Following varying numbers of days in culture, alkaline phosphatase (ALP) activity assays, ALP staining, alizarin red staining, ELISAs, and reverse transcription?quantitative polymerase chain reaction (RT?qPCR) and western blot analyses were performed to determine the osteogenic differentiation ability of hPDLSCs; RT?qPCR, western blot analysis, and immunofluorescence staining were conducted to investigate the underlying mechanisms. The canonical Wnt/??catenin pathway inhibitor XAV?939 and agonist CHIR?99021 were used to determine the contribution of the canonical Wnt/??catenin pathway to differentiation. Treatment with AGEs resulted in reduced ALP activity and Collagen I protein levels, decreased ALP staining, fewer mineralized nodules, and downregulated expression of osteogenic?specific genes [Runt?related transcription factor 2 (Runx2), Osterix, ALP, osteopontin (OPN), Collagen I and osteocalcin (OCN)] and proteins (Runx2, OPN, BSP and OCN); however, BBR partially rescued the AGE?induced decrease in the osteogenic potential of hPDLSCs. Furthermore, AGEs activated the canonical Wnt/??catenin signaling pathway and promoted the nuclear translocation of ??catenin; BBR partially attenuated this effect. In addition, XAV?939 partially rescued the AGE?induced reduction in the osteogenic potential of hPDLSCs, whereas CHIR?99021 suppressed the BBR?induced increase in the osteogenic potential of hPDLSCs. The present study indicated that AGEs attenuated the osteogenic differentiation ability of hPDLSCs, in part by activating the canonical Wnt/??catenin pathway; however, BBR attenuated these effects by inhibiting the canonical Wnt/??catenin pathway. These findings suggest a role for BBR in periodontal regeneration induced by hPDLSCs in patients with diabetes mellitus.
Project description:The anti-cancer activities of berberine (BBR) have been reported extensively in various cancer cell lines. However, the minimal inhibitory concentrations of BBR varied greatly among different cell lines and very few studies have been devoted to elucidate this aspect. In this study, we employed three cancer cell lines, HepG2, HeLa and SY5Y, to compare the transportation and distribution of BBR. HPLC results demonstrated that BBR was capable of penetrating all the cell lines whereas the cumulative concentrations were significantly different. HepG2 cells accumulated higher level of BBR for longer duration than the other two cell lines. Molecular docking studies revealed the BBR binding site on P-glycoprotein 1 (P-gp). In addition, we elucidated that BBR regulated P-gp at both mRNA and protein levels. BBR induced the transcription and translation of P-gp in HeLa and SY5Y cells, whereas BBR inhibited P-gp expression in HepG2 cells. Further study showed that BBR regulates P-gp expression depending on different mechanisms (or affected by different factors) in different cell lines. To summarize, our study has revealed several mechanistic aspects of BBR regulation on P-gp in different cancer cell lines and might shed some useful insights into the use of BBR in the anti-cancer drug development.
Project description:Berberine (BBR) has a neuroprotective effect against ischemic stroke, but its specific protective mechanism has not been clearly elaborated. This study explored the effect of BBR on the canopy FGF signaling regulator 2 (CNPY2) signaling pathway in the ischemic penumbra of rats. The model of cerebral ischemia-reperfusion injury (CIRI) was established by the thread embolization method, and BBR was gastrically perfused for 48 h or 24 h before operation and 6 h after operation. The rats were randomly divided into four groups: the Sham group, BBR group, CIRI group, and CIRI + BBR group. After 2 h of ischemia, followed by 24 h of reperfusion, we confirmed the neurologic dysfunction and apoptosis induced by CIRI in rats (<i>p</i> < 0.05). In the ischemic penumbra, the expression levels of CNPY2-regulated endoplasmic reticulum stress-induced apoptosis proteins (CNPY2, glucose-regulated protein 78 (GRP78), double-stranded RNA-activated protein kinase-like ER kinase (PERK), C/EBP homologous protein (CHOP), and Caspase-3) were significantly increased, but these levels were decreased after BBR treatment (<i>p</i> < 0.05). To further verify the inhibitory effect of BBR on CIRI-induced neuronal apoptosis, we added an endoplasmic reticulum-specific agonist and a PERK inhibitor to the treatment. BBR was shown to significantly inhibit the expression of apoptotic proteins induced by endoplasmic reticulum stress agonist, while the PERK inhibitor partially reversed the ability of BBR to inhibit apoptotic protein (<i>p</i> < 0.05). These results confirm that berberine may inhibit CIRI-induced neuronal apoptosis by downregulating the CNPY2 signaling pathway, thereby exerting a neuroprotective effect.