Resistant HepG2 Cells Research Articles

Berberine Ameliorates High-fat-induced Insulin Resistance in HepG2 Cells by Modulating PPARs Signaling Pathway.

Berberine (BBR), also known as berberine hydrochloride, was isolated from the rhizomes of the Coptis chinensis. Studies have reported that BBR plays an important role in glycolipid metabolism, including insulin (IR). The targets, and molecular mechanisms of BBR against hyperlipid-induced IR is worthy to be further studied. The related targets of BBR were identified via Pharmmapper database and relevant targets of diabetes were obtained through GeneCards and Online Mendelian Inheritance in Man (OMIM) database. The common targets were employed with the STRING database and visualized with the protein-protein interactions (PPI) network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to explore the biological progress and pathways. In vitro, human hepatocellular carcinomas (HepG2) cell was used as experimental cell line, and an insulin resistant HepG2 cell model (IR-HepG2) was constructed using free fatty acid induction. After intervention with BBR, glucose consumption and uptake in HepG2 cells were observed. Molecular docking was used to test the interaction between BBR and key targets, and real-time fluorescence quantitative PCR was used to detect the regulatory effect of BBR on related targets. 262 overlapped targets were extracted from BBR and diabetes. In the KEGG enrichment analysis, the peroxisome proliferator activated receptor (PPAR) signaling pathway was included. In vitro experiments, BBR can significantly increase sugar consumption and uptake in IR HepG2 cells, while PPAR inhibitors can weaken the effect of BBR on IR-HepG2. The PPAR signaling pathway is one of the important pathways for BBR to improve high-fat-induced insulin resistance in HepG2 cells.

Evaluation of cadmium effects on the glucose metabolism on insulin resistance HepG2 cells

Cadmium (Cd) is an environmental endocrine disruptor. Despite increasing research about the metabolic effects of Cd on HepG2 cells, information about the metabolic effects of Cd on insulin resistance HepG2 (IR-HepG2) cells is limited. Currently, most individuals with diabetes are exposed to Cd due to pollution. Previously, we reported that Cd exposure resulted in decreased blood glucose levels in diabetic mice, the underlying mechanism deserves further study. Therefore, we used palmitic acid (0.25 mM) to treat HepG2 cells to establish IR-HepG2 model. IR-HepG2 cells were exposed to CdCl2 (1 μM and 2 μM). Commercial kits were used to measure glucose production, glucose consumption, ROS and mitochondrial membrane potential. Western blot and qRT-PCR were used to measure the proteins and genes of glucose metabolism. In the current study setting, we found no significant changes in glucose metabolism in Cd-exposed HepG2 cells, but Cd enhanced glucose uptake, inhibited gluconeogenesis and activated the insulin signaling pathway in IR-HepG2 cells. Meanwhile, we observed that Cd caused oxidative stress and increased the intracellular calcium concentration and inhibited mitochondrial membrane potential in IR-HepG2 cells. Cd compensatingly increased glycolysis in IR-HepG2 cells. Collectively, we found Cd ameliorated glucose metabolism disorders in IR-HepG2 cells. Furthermore, Cd exacerbated mitochondrial damage and compensatory increased glycolysis in IR-HepG2 cells. These findings will provide novel insights for Cd exposure in insulin resistant individuals.

Nine new nor-3,4-seco-dammarane triterpenoids from the leaves of Cyclocarya paliurus and their hypoglycemic activity

This manuscript describes the isolation of nine new nor-3,4-seco-dammarane triterpenoids, norqingqianliusus A-I (1–9) and one known nortriterpenoid (10) from Cyclocarya paliurus leaves. Norqingqianliusus A and B (1 and 2) possess a unique 3,4-seco-dammarane-type C26 tetranortriterpenoid skeleton. The compounds were structurally characterized through modern spectroscopic techniques. Moreover, the potential mechanism of hypoglycemic activity was further explored by studying the effects on glucosamine-induced insulin resistant HepG2 cells. In vitro hypoglycemic effects of all of the isolates were investigated using insulin resistant HepG2 cells. The glucose consumption was significantly promoted by compound 10, in a dose-dependent manner, thus alleviating damage in IR-HepG2 cells. Besides, it reduced the PEPCK and GSK3β gene expression, involved in glucose metabolism. The anti-diabetic effects of the plant, utilized traditionally, can hence be attributed to the presence of nor-3,4-seco-dammarane triterpenoids in the leaves.

Improvement of glucolipid metabolism and oxidative stress via modulating PI3K/Akt pathway in insulin resistance HepG2 cells by chickpea flavonoids

Chickpea (Cicer arietinum L.) is a significant dietary source of flavonoids and the hypoglycemic activity were investigated in this study. Firstly, total twenty nine chickpea flavonoids were identified by UPLC-MS/MS with ononin, cyanidin-3-O-glucoside, astragalin, cynaroside, kaempferol-3-O-rutinoside, biochanin A, and daidzin being the most abundant among them. Our results demonstrated that chickpea flavonoids regulated glucose metabolism and lipid metabolism, and reduced oxidative stress in insulin resistance HepG2 cells. Furthermore, insulin resistance was ameliorated by chickpea flavonoids through the activation of insulin receptor substrate1 (IRS1), phosphoinositide 3-kinase (PI3K), and phosphorylated protein kinase B (Akt) in HepG2 cells. More importantly, key differential metabolites include L-tryptophan, L-tyrosine, l-glutamine and linoleic acid were reserved by chickpea flavonoids and correlated with glucolipid metabolism and oxidative stress in IR-HepG2 cells. In conclusion, these results indicated that chickpea flavonoids might act as potential natural products regulating insulin resistance in HepG2 cells.

Discovery and identification of natural alkaloids with potential to impact insulin resistance syndrome in Cyclocarya paliurus. (Batal) leaves by UPLC-QTOF-MS combined with HepG2 cells

Cyclocarya paliurus (Batal.) leaves, which contain a range of bioactive compounds, have been used as a traditional Chinese medicine homologous food since ancient times. However, there is a paucity of literature on comprehensive studies of alkaloids in the leaves of Cyclocarya paliurus (Batal.). For the first time, this study aimed to discover and identify alkaloids extracted from Cyclocarya paliurus (Batal.) leaves by ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-QTOF-MS). A total of ten alkaloids have been identified from Cyclocarya paliurus (Batal.) leaves based on accurate mass spectra (mass accuracy, isotopic spacing and distribution) and comparison to fragmentation spectra reported in the literature. In vitro, alkaloids alleviated insulin resistance by increasing glucose consumption and glycogen content in insulin resistance HepG2 cells. The RNA-seq and western blotting results showed that alkaloids could upregulate the expression of phosphatidylinositol 3-kinase (PI3K), and increase the phosphorylation of insulin receptor protein kinase B (AKT). This study not only clarified the chemical constituents and revealed that diverse alkaloids also presented from Cyclocarya paliurus (Batal.) leaves, also, it will provide chemical information on potential compounds for developing new drugs.

Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells

ObjectiveResearch has shown that celastrol can effectively treat a variety of diseases, yet when passing a certain dosage threshold, celastrol becomes toxic, causing complications such as liver and kidney damage and erythrocytopenia, among others. With this dichotomy in mind, it is extremely important to find ways to preserve celastrol’s efficacy while reducing or preventing its toxicity. MethodsIn this study, insulin-resistant HepG2 (IR-HepG2) cells were prepared using palmitic acid and used for in vitro experiments. IR-HepG2 cells were treated with celastrol alone or in combination with N-acetylcysteine (NAC) or ferrostatin-1 (Fer-1) for 12, 24 or 48 h, at a range of doses. Cell counting kit-8 assay, Western blotting, quantitative reverse transcription-polymerase chain reaction, glucose consumption assessment, and flow cytometry were performed to measure celastrol’s cytotoxicity and whether the cell death was linked to ferroptosis. ResultsCelastrol treatment increased lipid oxidation and decreased expression of anti-ferroptosis proteins in IR-HepG2 cells. Celastrol downregulated glutathione peroxidase 4 (GPX4) mRNA. Molecular docking models predicted that solute carrier family 7 member 11 (SLC7A11) and GPX4 were covalently bound by celastrol. Importantly, we found for the first time that the application of ferroptosis inhibitors (especially NAC) was able to reduce celastrol’s toxicity while preserving its ability to improve insulin sensitivity in IR-HepG2 cells. ConclusionOne potential mechanism of celastrol’s cytotoxicity is the induction of ferroptosis, which can be alleviated by treatment with ferroptosis inhibitors. These findings provide a new strategy to block celastrol’s toxicity while preserving its therapeutic effects.Please cite this article as: Liu JJ, Zhang X, Qi MM, Chi YB, Cai BL, Peng B, Zhang DH. Ferroptosis inhibitors reduce celastrol toxicity and preserve its insulin sensitizing effects in insulin resistant HepG2 cells. J Integr Med. 2024; 22(3): 286–294.

Antihyperglycemic effects of triterpenoid saponins from the seeds of Aesculus chinensis Bge

Six undescribed triterpenoid saponins, namely aescuchinosides A-F, along with seven known triterpenoid saponins, were isolated from the seeds of Aesculus chinensis. Barrigenol-like triterpenoids (BATs) constitute these saponins. Protoaescigenin serves as their aglycone, with various oxygen-containing groups, including acetyl, isobutyryl, tigloyl, and angeloyl groups situated at C-21, C-22, and C-28. Various techniques, including 1D and 2D-NMR spectroscopy, high-resolution mass spectrometry, and acid hydrolysis, were employed to determine the structures of these compounds. The antihyperglycemic effects of the isolated compounds were examined in insulin -resistant HepG2 cells induced by palmitic acid treatment. At a concentration of 6 μM, aesculinoside F exhibited a significant increase in glucose consumption. In addition, aesculinoside F demonstrated the potential to improve insulin resistant by upregulating the PI3K/AKT pathway. These results indicate that the seeds ofA.chinensis hold promising potential for preventing insulin resistant related disease.

Secondary metabolites from Scindapsus officinalis (Roxb.) Schott. with in vitro antidiabetic activities

One new phenolic cyclobutantetraol ester united chromone glycoside (1), one new amide (2), and three new phenyl ethanol glycosides (3–5) were obtained from the water extract of Scindapsus officinalis (Roxb.) Schott, in which compound 1 was the first reported structure incorporating the phenolic cyclobutantetraol ester and chromone via the glucose phenolic metabolites in nature. Structures of the isolated compounds, including absolute configurations, were elucidated according to the analysis of HRESIMS, NMR, ECD and BLYP/6-31G* geometry optimization calculations of 13C NMR data. All isolates (1–5) were evaluated for the antidiabetic activity by the insulin resistance (IR) model and anti-inflammatory activity against NO production in vitro. Compounds 1–3 showed strong antidiabetic activities, greatly promoting the glucose consumption in the insulin resistance HepG2 cells compared with the model group, however, 1–5 showed weak anti-inflammatory activities.

Horizontal transfer of miR-383 sensitise cells to cisplatin by targeting VEGFA-Akt signalling loop.

Cellular resistance to cisplatin has been one of the major obstacles in the success of combination therapy for many types of cancers. Emerging evidences suggest that exosomes released by drug resistant tumour cells play significant role in conferring resistance to drug sensitive cells by means of horizontal transfer of genetic materials such as miRNAs. Though exosomal miRNAs have been reported to confer drug resistance, the exact underlying mechanisms are still unclear. In the present study, mature miRNAs secreted differentially by cisplatin resistant and cisplatin sensitive HepG2 cells were profiled and the effect of most significantly lowered miRNA in conferring cisplatin resistance when horizontally transferred, was analysed. we report miR-383 to be present at the lowest levels among the differentially abundant miRNAs expressed in exosomes secreted by cisplatin resistant cells compared to that that of cisplatin sensitive cells. We therefore, checked the effect of ectopic expression of miR-383 in altering cisplatin sensitivity of Hela cells. Drug sensitivity assay and apoptotic assays revealed that miR-383 could sensitise cells to cisplatin by targeting VEGF and its downstream Akt mediated pathway. Results presented here provide evidence for the important role of miR-383 in regulating cisplatin sensitivity by modulating VEGF signalling loop upon horizontal transfer across different cell types.

Anti-Hyperglycemic Effect of the Brown Slime Cap Mushroom Chroogomphus rutilus (Agaricomycetes) Crude Polysaccharide In Vitro and In Vivo.

The prevalence of diabetes is increasing worldwide, and it is very important to study new hypoglycemic active substances. In this study, we investigated the hypoglycemic effect of Chroogomphus rutilus crude polysaccharide (CRCP) in HepG2 cells and streptozotocin-induced diabetic mice. A glucose consumption experiment conducted in HepG2 cells demonstrated the in vitro hypoglycemic activity of CRCP. Furthermore, CRCP exhibited significant hypoglycemic effects and effectively ameliorated insulin resistance in insulin resistant HepG2 cells. In high-fat diet and streptozotocin-induced diabetic mice, after 4 weeks of CRCP administration, fasting blood glucose, fasting serum insulin, triglyceride, total cholesterol, low-density lipoprotein cholesterol, glutamate transaminase, alanine transaminase, and insulin resistance index significantly decreased, while high-density lipoprotein cholesterol and insulin sensitivity index (ISI) were markedly increased. Moreover, hematoxylin-eosin (HE) staining and immunofluorescence labeling of tissue sections indicated that CRCP attenuated the pathological damage of liver and pancreas in diabetic mice. These results indicate that CRCP is a potential hypoglycemic agent.

Baicalin ameliorates insulin resistance and regulates hepatic glucose metabolism via activating insulin signaling pathway in obese pre-diabetic mice

BackgroundDiabetes belongs to the most prevalent metabolic diseases worldwide, which is featured with insulin resistance, closely associated with obesity and urgently needs to be treated. Baicalin, belonging to natural flavonoids, has been reported to inhibit oxidative stress or inflammatoin. PurposeThis study investigated the properties of baicalin on modulating abnormal glucolipid metabolism, as well as the underlying in-vitro and in-vivo mechanisms. MethodsInsulin-resistant (IR)-HepG2 cells were stimulated by dexamethasone (20 µM) and high glucose (50 mM) for 48 h and incubated with or without baicalin or metformin for another 16 h. Male C57BL/6 J mice were fed with a high-fat diet (HFD, 60 % kcal% fat) during the total 14 weeks. Obese mice were then administered with baicalin (50 and 100 mg/kg) or vehicle solution everyday through oral gavage during the last 4-week period. Moreover, baicalin metabolisms in vitro and in vivo were determined using UPLC/MS/MS to study its metabolism situation. ResultsExposure to dexamethasone and high glucose damaged the abilities of glycogen synthesis and glucose uptake with elevated oxidative stress and increased generation levels of advanced glycation end-products (AGEs) in HepG2 cells. These impairments were basically reversed by baicalin treatment. Four-week oral administration with baicalin ameliorated hyperglycemia and dyslipidemia in HFD-induced obese and pre-diabetic mice. Downregulation of IRS/PI3K/Akt signaling pathway accomplished with reduced GLUT4 expression and enhanced GSK-3β activity was observed in insulin resistant HepG2 cells as well as liver tissues from pre-diabetic mice; and such effect was prevented by baicalin. Moreover, baicalin and its matabolites were detected in IR-HepG2 cells and mouse plasma. ConclusionThe study illustrated that baicalin alleviated insulin resistance by activating insulin signaling pathways and inhibiting oxidative stress and AGEs production, revealing the potential of baicalin to be a therapeutic natural flavonoid against hepatic insulin and glucose-lipid metabolic disturbance in pre-diabetes accompanied with obesity.

Design, synthesis of novel chromene-based scaffolds targeting hepatocellular carcinoma: Cell cycle arrest, cytotoxic effect against resistant cancer cells, apoptosis induction, and c-Src inhibition.

New chromene derivatives were synthesized based on 4-(3,4-dimethoxy)-4H-chromene scaffold. All target compounds exhibited cytotoxic activity against HepG2 cells (IC50 = 2.40-141.22 μM). Chromens 5 and 9 showed superior cytotoxicity over staurosporine (IC50 = 18.27 μM) and vinblastine (IC50 = 5.20 μM). c-Src kinase inhibition assay of compounds 5 and 9 displayed the dominant c-Src inhibitory activity of 5 (IC50 = 0.184 μM) over 9 (IC50 = 0.288 μM). The safety of the most potent compound 5 against normal WI-38 cells was confirmed via its IC50 of 115.75 μM comparable with 5-FU (IC50 = 16.28 μM). Moreover, the promising chromene 5 displayed potent cytotoxicity against resistant HepG2 cells with IC50 of 26.03 μM comparable with 5-FU (IC50 = 42.68 μM). The most active chromene 5 arrested the HepG2 cell cycle at the S phase and induced a 29-fold increase in the total number of apoptotic cells indicating pre-G1 apoptosis. The ability of compound 5 to induce apoptosis was supported via elevation of caspase-3, caspase-7, caspase-9 and proapoptotic Bax protein levels in addition to downregulation of the antiapoptotic Bcl2 protein. Molecular docking studies of compound 5 showed good binding interaction pattern inside c-Src kinase enzyme active site.

Lactoferrin decorated bilosomes for the oral delivery of quercetin in type 2 diabetes: In vitro and in vivo appraisal

Despite its tremendous potential for type 2 diabetes management, quercetin (QRC) suffers poor gastric stability, poor bioavailability, and extensive first pass metabolism. Drug encapsulation into bilosomes (BSL) has proven enhanced properties in-vitro and in-vivo. Herein, this work endeavoured to evaluate efficacy of QRC-encapsulated bilosomes capped with lactoferrin (LF); a milk protein with antidiabetic potential, for type 2 diabetes oral treatment. The optimized formulation (LF-QRC-BSL) was evaluated in-vitro on α-amylase enzyme inhibition and insulin resistant HepG2 cell model and in vivo on streptozocin/high fat diet induced diabetes in rats. LF-QRC-BSL showed a small size (68.1 nm), a narrow PDI (0.18) and a −25.5 mV zeta potential. A high entrapment efficiency (94 %) with sustained release were also observed. LF-QRC-BSL displayed 100 % permeation through excised diabetic rat intestines after 6 h, 70.2 % inhibition of α-amylase enzyme in-vitro and an augmented recovery of glucose uptake in insulin resistant cells. In diabetic rats, LF-QRC-BSL resulted in significant decrease in blood glucose level, improved lipid profile and tissue injury markers with reduced oxidative stress and inflammatory markers. Further, histopathological examination of the kidneys, liver and pancreas revealed an almost restored normal condition comparable to the negative control. Overall, LF-QRC-BSL have proven to be a promising therapy for type 2 diabetes.

Necessity of Different Lignocellulose on Exopolysaccharide Synthesis and Its Hypoglycemic Activity In Vitro of Inonotus obliquus.

Current work proposes elm sawdust, poplar sawdust, pine sawdust, and cotton straw with different lignocellulose compositions and structures as the research objects to investigate the relationship between the hypoglycemic activity of mycelium polysaccharides from Inonotus obliquus and lignocellulose biodegradation. Four kinds of lignocellulosic materials could significantly increase the exopolysaccharide content and α-glucosidase inhibition rate and advance the occurrence time of α-glucosidase inhibition activity. Among all groups, the polysaccharide synthesis promotion effect of the cotton straw group was the best, which exopolysaccharide yield was 92.05% higher than that of the control group after 11-day fermentation. Meanwhile, the highest α-glucosidase inhibitory activity was found in the elm sawdust group on the 11th day (30.99%, which was 137.47% higher than control), and the exopolysaccharide in the elm sawdust group showed its effectiveness on glucose consumption of insulin resistant HepG2 cells at the concentration of 20 µg/mL, significantly higher than that of the metformin group (P < 0.05). The cellulose in the non-crystalline region of elm and pine and the hemicellulose of poplar were mainly used in the fermentation of I. obliquus, while the cellulose in the crystalline zone and amorphous zone of cotton straw was degraded to improve the exopolysaccharide content of I. obliquus. This paper revealed the necessity of different kinds of lignocellulose for the synthesis of active polysaccharide from I. obliquus and provided a new idea for the regulation of polysaccharide synthesis pathway.

Novel benzotriazole-benzimidazole metal complexes: structure-activity relationship, synthesis, characterization, and antidiabetic activity

To investigate the relationship between structure and antidiabetic activity, four novel metal complexes [Cu2(ebmb)2Cl4] (1), [Zn2(ebmb)Cl4] (2), [Cu(ebmb)SO4]n (3) and [Cu(mbmb)SO4]n (4) have been synthesized by 1-[(2-ethyl-1H-benzimidazole- 1-yl)methyl]-1H-benzotriazole (ebmb) and 1-[(2-methyl-1H-benzimidazole-1-yl) methyl]-1H-benzotriazole (mbmb), and the antidiabetic activities of them have been evaluated. The X-ray single crystal diffraction analysis reveals that complexes 1-2 are binuclear complexes and isostructural expect the different central metal. Complexes 3-4 possess the same central metal but different ligands, and they have very similar structure and they both possess one-dimensional chain structure. We assessed the in vitro antidiabetic activities of the complexes. The results of the α-amylase and α-glucosidase inhibition assays showed that complexes 1 and 4 possessed strongest inhibitory activities with the IC50 values of 1.29mM±0.04 (1) and 4.69mM±0.43 (4) for α-amylase, 0.75±0.04μM (1) and 1.63±0.11μM (4) for α-glucosidase. Finally, we evaluated the effect of complexes 1 and 4 on the glucose consumption of insulin resistant (IR) HepG2 cells, the results showed that both two complexes can improve the IR state of HepG2 cells, the glucose consumption of complex 1-treated IR-HepG2 cells and complex 4-treated IR-HepG2 cells were 1.3 times and 1.18 times as much as the model group, respectively. What's more, the structure-function relationship of complexes had also been discussed in this paper.

A polysaccharide NAP-3 from Naematelia aurantialba: Structural characterization and adjunctive hypoglycemic activity

A novel polysaccharide (NAP-3) was isolated and purified from Naematelia aurantialba after water extraction. The structure of NAP-3, which was determined by FT-IR, HPLC, GC–MS, and NMR, indicated that NAP-3 was a homogeneous polysaccharide with the molecular weight of 428 kDa, mainly consisted of β-1, 3-D-Manp, β-1, 2, 3-D-Manp, β-D-Xylp, β-1, 4-D-Glcp, β-1, 4-D-Rhap in a molar ratio of 6.49: 1.11: 2.4: 0.13: 0.83. In vitro α-glucosidase and α-amylase inhibitory assay showed that NAP-3 had a low IC50 value, which exhibited similar enzyme inhibitory activity as acarbose. NAP-3 was evaluated as an adjuvant with metformin for antidiabetic therapy in HFD/STZ-induced diabetic mice and insulin resistance HepG2 cells. The combination of NAP-3 and metformin in diabetic mice exhibited significant hypoglycemic activity, reducing body weight, serum insulin levels, glucose tolerance, insulin tolerance, and increasing antioxidant levels compared to metformin alone. The combination of NAP-3 and metformin improved oxidative stress by increasing ROS clearance, thereby enhancing glucose uptake in HepG2 cells. This study provided new data for the study of Naematelia aurantialba polysaccharides and offers a new adjuvant therapy for the treatment of diabetes.

The Hypoglycemic Activities and Underlying Mechanisms of Two Saponins-Rich Components from Fried Ziziphus jujuba Mill. Kernel.

Dried Ziziphus jujuba Mill. kernel is a potential natural source of nutraceutical and therapeutic agents in China. Recent researches have shown that the saponins of dried Z. jujuba Mill. kernel (SZJs: SZJ-1 and SZJ-2) have various biological effects. However, the hypoglycemic activities and underlying mechanisms of SZJs remain obscure. In the current study, two saponins SZJ-1 and SZJ-2 mainly composed of betulinic acid, spinosin, jujuboside A, and jujuboside B are extracted and is olated from dried Z. jujuba Mill. kernel. The SZJ-1 and SZJ-2 could significantly inhibit the activities of digestion enzymes α-glucosidase and α-amylases. The hypoglycemic ability of SZJ-1 and SZJ-2 is further investigated and the results show that SZJ-1 and SZJ-2 can improve the hyperglycemic by increasing the glucose consumption, improving the superoxide dismutase (SOD), hexokinase (HK), pyruvate kinase (PK) activities, and decrease the MDA content of insulin resistant HepG2 cells. Furthermore, SZJ-1 and SZJ-2 can activate the phosphorated adenosine 5'-monophosphate (AMP)-activated protein kinase α (p-AMPK), phosphoinositide 3-kinase p110α (PI3K-p110α), and phosphorated glycogen synthase kinase-3β (Ser9) (p-GSK3β). These resultsindicating that the SZJ-1 and SZJ-2 might improve the insulin resistant symptoms by improving the energy metabolic level and increasing the glycogen synthase activity of HepG2 cells.

Phytochemical profiling, in vitro antioxidants, and antidiabetic efficacy of ethyl acetate fraction of Lespedeza cuneata on streptozotocin-induced diabetic rats.

In the recent past, phytomolecules are exponentially applied in discovering the antidiabetic drug due to less adverse effects. This work screened the active solvent fraction of Lespedeza cuneata based on the phytochemical, enzyme inhibition, and antioxidant properties. The antioxidant efficacy of the different fractions of the L. cuneata was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing power, hydrogen peroxide, and hydroxyl radical scavenging assays. The digestiveenzyme (α-amylase and α-glucosidase) inhibitory activity was also evaluated. The phytochemical composition of ethyl acetate fraction of L. cuneata (Lc-EAF) was studied by UHPLC-QTOF-MS/MS. The effect of Lc-EAF treatments on glucose uptake was studied in insulin resistance HepG2 cells (IR-HepG2). Further, the antidiabetic effect of Lc-EAF in streptozotocin (STZ)-induced diabetic mice were demonstrated. Ethyl acetate, hexane, and methanol fractions of the L. cuneata showed notable antioxidant, α-amylase, and α-glucosidase inhibitory properties. Among the fractions, Lc-EAF was found to be the most potent. The Lc-EAF exhibited an IC50 of 205.32 ± 23.47µg/mL and 105.32 ± 13.93µg/mL for α-amylase and α-glucosidase inhibition, respectively. In addition, 75µg/mL of Lc-EAF exposure enhanced glucose uptake (68.23%) in IR-HepG2 cells. In vivo study indicated that treatmentof Lc-EAF (100mg/kg b.wt) maintained the blood glucose level through reduced insulin level while improving the lipid profile, hepatic, and renal markers. These findings suggest that Lc-EAF could be considered a prominent sourcefor antidiabetic, anti-hyperlipidemic, and anti-ROS potentials.

A rapid and sensitive single-cell proteomic method based on fast liquid-chromatography separation, retention time prediction and MS1-only acquisition

Single-cell analysis has received much attention in recent years for elucidating the widely existing cellular heterogeneity in biological systems. However, the ability to measure the proteome in single cells is still far behind that of transcriptomics due to the lack of sensitive and high-throughput mass spectrometry methods. Herein, we report an integrated strategy termed “SCP-MS1” that combines fast liquid chromatography (LC) separation, deep learning-based retention time (RT) prediction and MS1-only acquisition for rapid and sensitive single-cell proteome analysis. In SCP-MS1, the peptides were identified via four-dimensional MS1 feature (m/z, RT, charge and FAIMS CV) matching, therefore relieving MS acquisition from the time consuming and information losing MS2 step and making this method particularly compatible with fast LC separation. By completely omitting the MS2 step, all the MS analysis time was utilized for MS1 acquisition in SCP-MS1 and therefore led to 65%–138% increased MS1 feature collection. Unlike “match between run” methods that still needed MS2 information for RT alignment, SCP-MS1 used deep learning-based RT prediction to transfer the measured RTs in long gradient bulk analyses to short gradient single cell analyses, which was the key step to enhance both identification scale and matching accuracy. Using this strategy, more than 2000 proteins were obtained from 0.2 ng of peptides with a 14-min active gradient at a false discovery rate (FDR) of 0.8%. Comparing with the DDA method, improved quantitative performance was also observed for SCP-MS1 with approximately 50% decreased median coefficient of variation of quantified proteins. For single-cell analysis, 1715 ± 204 and 1604 ± 224 proteins were quantified in single 293T and HeLa cells, respectively. Finally, SCP-MS1 was applied to single-cell proteome analysis of sorafenib resistant and non-resistant HepG2 cells and revealed clear cellular heterogeneity in the resistant population that may be masked in bulk studies.

Bacterial metabolite butyrate in modulating sorafenib-targeted microRNAs to curtail its resistance in hepatocellular carcinoma.

The host dietary fibre is fermented into short-chain fatty acids (SCFA) by intestinal microbiota as bacterial metabolites like propionate, acetate and butyrate. Among these metabolites, the role of butyrate is well documented to provide energy to intestinal epithelial cells. Also, butyrate has anti-inflammatory and anti-tumour properties and decrease in its level by unbalanced diet can develops cancer. Lately, some research has suggested that sodium butyrate as an inhibitor of histone deacetylase (HDAC) may have anticancer potential for hepatocellular carcinoma (HCC), the most common type of liver cancer. Since, HCC is asymptomatic it is usually diagnosed at its advanced stage. Sorafenib with antiproliferative and antiangiogenic effects is the first line of treatment in advanced HCC. However, prolonged drug treatment to HCC patients develops adaptive resistance towards the sorafenib. Sorafenib resistance can also be enhanced by differentially expressed microRNAs. However, the significance of butyrate in HCC sorafenib resistance and its association with sorafenib-targeted microRNAs is yet to be unfurled. Here, an attempt has been made to explore the role of bacterial metabolite butyrate on sorafenib resistant HCC as well as on sorafenib-targeted microRNAs (miR-7641 and miR-199) to curtail sorafenib resistance in HCC. Initially, in-silico analysis was performed using Human Metabolome Database (HMDB) so to identify specific butyrate producing faecal bacteria. Then, their specific 16s rRNA expression was compared between HCC patients and healthy individuals using qRT-PCR. Additionally, the cell viability (MTT) and apoptosis assays were performed in both parental and sorafenib resistant HepG2 cells to evaluate the role of sodium butyrate in sorafenib resistant HCC. Moreover, the association of sodium butyrate with sorafenib-targeted miR-7641 and miR-199 was also assessed using real time PCR, cell viability, cell apoptosis and transfection assays. In silico analysis demonstrated Roseburia cecical, Roseburia intestinalis, Eubacterium rectal, Faecalibacterium prausnitzii as specific butyrate producing faecal bacteria and their 16s rRNA expression was downregulated in HCC patients. In vitro study revealed the presence of sodium butyrate also decreased the cell viability as well as enhanced cell apoptosis of both parental and resistant HepG2 cells. Interestingly, sodium butyrate also decreased the expression of both sorafenib-targeted miR-7641 and miR-199. Further, combination of both sodium butyrate and antimiR-7641 or antimiR-199 also increased apoptosis and decreased viability of resistant cells. This is first study to unravel the association of butyrate producing bacteria with HCC patients and the significance of bacterial metabolite butyrate as anti-tumour in sorafenib resistant hepatocellular carcinoma. The study also demonstrated the plausible new aspects of bacterial metabolite butyrate association with sorafenib-targeted miRNAs (miR-7641 and miR-199). Hence, the study highlighted the therapeutic potential of bacterial metabolite butyrate that might improve the clinical management of hepatocellular carcinoma.