Main Research Findings

1) Treatment with Vincamine was shown to improve glucose homeostasis and protect beta-cell function in a model of type 2 diabetic mice.

2) Synthetic derivative of Vincamine, Vinpocetine, was found to protect against osteoarthritis by inhibiting degradation of the extracellular matrix and inhibiting ferroptosis.

 

Selected Data

1) The research performed by Du et al aimed to investigate Vincamine’s potential as a GPR40 agonist, evaluating its capacity to improve glucose homeostasis and protect β-cell function in both in vitro and in vivo models of type 2 diabetes mellitus (T2DM). For in vitro investigations, the study utilized INS-832/13 rat insulinoma cells and hGPR40-CHO cells. INS-832/13 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin/streptomycin, L-glutamine, sodium-pyruvate, and β-mercaptoethanol. hGPR40-CHO cells were maintained in F12 medium with similar supplements and G418 for selection. Mouse primary hepatocytes were isolated using a perfusion method involving EGTA-containing PBS and collagenase-supplemented DMEM, followed by filtration and centrifugation to harvest fresh hepatocytes [1].

To assess cell health and function, various assays were employed. Cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay, where INS-832/13 cells were treated with streptozotocin (STZ, 0.4 mM) to induce damage, and Vincamine in doses ranging from 0.1–20 µM, was tested for its protective effects. Cell proliferation was measured using the CyQUANT Cell Proliferation Assay Kit. Apoptosis was quantified via flow cytometry, staining cells with FITC Annexin V-FITC/PI after STZ and Vincamine treatments, with 10,000 events recorded per sample. Caspase 3 and Caspase 9 activities were measured using Apo-ONE Homogeneous Caspase-3/7 Assay and Caspase-Glo 9 Assay kits, respectively.

Signaling pathway components were analyzed using several techniques. The p-Akt AlphaLISA assay, a bead-based immunoassay, was used to quantify phosphorylated Akt levels in cell lysates. Total RNA was extracted using RNAiso Plus, reverse-transcribed into cDNA, and gene expression levels of Irs2, p21, Bim, Gpr40, and Gapdh were quantified by quantitative real-time PCR using SYBR Premix Ex Taq and specific primers. Protein expression was examined by Western blot assay: total proteins from cells or tissues were separated by SDS-PAGE, transferred to nitrocellulose membranes, incubated with specific primary antibodies and secondary antibodies, and detected using specific software systems [1].

Immunohistochemistry (IHC) assays were performed on pancreatic tissues to visually assess the protection of β-cells and the overall structure of islets. Tissue slides, prepared from paraffin-embedded pancreases, underwent standard IHC procedures, including antigen retrieval in the citrate buffer. Insulin-positive signals were visualized to quantify the area of pancreatic islets, with intensity measured using Image-Pro software. For specific protein visualization and localization, immunofluorescence (IF) assays were conducted to detect IRS2, cleaved Caspase 3, and insulin. After deparaffination and rehydration, sections were incubated with primary antibodies overnight at 4°C, followed by secondary antibody incubation at room temperature. DAPI was used for counterstaining, and images were captured using a microscope under consistent settings.

Functional cellular assays were critical to understand Vincamine’s impact on glucose metabolism. Glucose-stimulated insulin secretion (GSIS) assays were carried out in INS-832/13 cells. Cells were pre-incubated in Krebs-Ringer bicarbonate buffer containing 0.2% BSA for 2 hours, then incubated for another 2 hours with either 2.8mM (low) or 16.8mM (high) glucose, along with test compounds like Vincamine or glibenclamide (as a positive control). Insulin content in the supernatant was subsequently measured using the AlphaLISA insulin kit. Enzymatic activities of key metabolic regulators were also assessed. PI3K activity was measured using a specific PI3K activity ELISA kit. Glucokinase (GK) activity was detected by monitoring NADH production at 340nm using FlexStation 3, as previously described. PDE activity was measured using a commercial PDE Activity Assay Kit [1].

The study also investigated Vincamine’s direct interaction with GPR40. GPR40 activity was assessed using a fluorescence imaging plate reader (FLIPR) assay in hGPR40-CHO cells. This method continuously monitored cytosolic Ca2+ signals (494nm/525nm fluorescence) after pre-incubation with Fluo-4 AM. The change in Ca2+ flow upon addition of test compounds was regarded as a measure of GPR40 activation. A Cellular Thermal Shift Assay (CETSA) was employed to confirm direct binding of Vincamine to GPR40. INS-832/13 cells were homogenized, treated with Vincamine, and then subjected to a series of gradient temperatures. The thermal stability of GPR40 and other control proteins (Akt, GSK3β, GAPDH) was assessed by Western blot analysis of soluble protein fractions, with TAK-875 serving as a positive control.

To further dissect molecular pathways, specific genetic manipulations were performed. siRNA transfection was used to knock down GPR40 expression in INS-832/13 cells, utilizing Lipofectamine RNAIMAX and non-targeting siRNA as a negative control. Gene expression levels were verified by qRT-PCR and Western blot. Additionally, a GPR40-overexpression plasmid was constructed by inserting GPR40-cDNA into a p3xFLAG-myc-CMV-24 vector and transiently transfected into INS-832/13 cells using Lipofectamine 3000, with an empty vector as a negative control [1].

For in vivo efficacy, two diabetic mouse models were used: HFD/STZ (high-fat diet fed and streptozotocin-induced) and db/db mice as a control. HFD/STZ mice were generated by feeding a 58% fat diet for 4 weeks, followed by a single intraperitoneal (i.p.) injection of 100mg/kg STZ. Diabetic mice were screened by 6-hour fasting blood glucose levels and randomly assigned to groups. Vincamine hydrochloride in a dose of 15 or 30mg/kg/day was administered daily via i.p. injection for 5–6 weeks. Fasting blood glucose, HbA1c (glycated hemoglobin), oral glucose tolerance tests (OGTT), and insulin tolerance tests (ITT) were monitored weekly or at specific time points. Blood glucose and insulin levels were measured from tail vein samples, with insulin content determined by AlphaLISA. Diet and body weight were monitored throughout [1].

2) This study conducted by the research team of Wang et al aimed to elucidate the protective effects and underlying mechanisms of Vinpocetine (Vin) against osteoarthritis (OA) by targeting ferroptosis and extracellular matrix (ECM) degradation, primarily through the Nrf2/GPX4 pathway. The research integrated both in vitro and in vivo models, supported by advanced computational and analytical techniques [2].

For in vitro studies, primary chondrocytes were extracted from the knee cartilage of neonatal C57BL/6 mice aged 3-5 days old. Cartilage tissue was carefully dissected, rinsed with PBS, and enzymatically digested with 2 mg/ml collagenase II for 4 hours at 37°C. The isolated cells were then cultured in DMEM/F12 medium supplemented with 1% penicillin-streptomycin and 10% FBS at 37°C in a 5% CO2 incubator. Second-passage cells were used for all experiments, with daily medium changes. Cell viability was assessed using the CCK-8 assay, where chondrocytes were seeded in 96-well plates and exposed to various concentrations of Vin ranging from 0–160 µM for 1-2 days, followed by CCK-8 reagent incubation and absorbance measurement at 450 nm. Cytotoxicity and ferroptosis induction were simulated by exposing chondrocytes to 50 µM tert-butyl hydroperoxide (TBHP), with 5 µM Erastin as a positive ferroptosis inducer and 1 µM Ferrostatin-1 (Fer-1) as an inhibitor. Lactate dehydrogenase (LDH) release was measured using an LDH assay kit, and absorbance was read at 490 nm.

Oxidative stress and lipid peroxidation were quantified using specific detection kits. After thorough washing, chondrocytes were lysed, and glutathione (GSH) and malondialdehyde (MDA) activities were measured using commercial kits from Sigma. Intracellular ferrous ions (Fe2+) levels, indicative of iron overload in ferroptosis, were assessed using an Abcam iron assay kit. Morphological changes in chondrocytes and ECM were visualized through Toluidine blue (TB) and Safranin O (SO) staining. Cells were fixed with 4% paraformaldehyde, stained with TB and SO, and observed under an Olympus microscope. Lipid peroxidation was further evaluated using BODIPY-C11 staining, and intracellular Fe2+ levels were also visualized with FerroOrange staining [2].

Further, intracellular reactive oxygen species (ROS) levels were detected using fluorescence probes dihydroethidium (DHE) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) from Invitrogen and Beyotime, respectively. Cells were rinsed, exposed to probes, and observed under an Olympus microscope, with detailed analysis performed using Image ProPlus software. Mitochondrial morphology and function were assessed using transmission electron microscopy (TEM). Chondrocytes were fixed, stained with osmium tetroxide and uranyl acetate, dehydrated, embedded in Araldite epoxy resin, sliced, and observed under a Hitachi Field Emission TEM. Mitochondrial ROS levels and membrane potential were evaluated using fluorescent probes JC-1 and MitoSox, while MitoTracker Red was used to visualize mitochondrial distribution and shape via laser scanning confocal microscopy [2].

Molecular and biochemical analyses were crucial for deciphering signaling pathways. Western blotting was performed to quantify protein levels. Total proteins from chondrocytes or tissues were extracted using a RIPA lysis buffer containing phosphatase and protease inhibitors. Samples were denatured, separated by SDS-PAGE, blotted onto PVDF membranes, and incubated with primary antibodies against key proteins such as GPX4, SLC7A11, ACSL4, Opal, Drp1, Mfn1, Mfn2, HO-1, Nrf2, Keap1, NQO1, collagen II, aggrecan, MMP-13, ADAMTS-5, and GAPDH. After washing, membranes were incubated with secondary antibodies, and detection was performed using enhanced chemiluminescence with a Bio-Rad ChemiDoc XRS+ imaging system. Immunoprecipitation assays were conducted to investigate the interaction between Nrf2 and Keap1. Chondrocyte protein lysates were incubated with an anti-Nrf2 primary antibody, immune complexes were enriched using magnetic beads, and subsequent Western blotting with an anti-Keap1 antibody was performed. Immunofluorescence techniques further confirmed protein expression and localization for collagen II, MMP-13, SLC7A11, GPX4, and Nrf2 in chondrocytes, with staining intensity quantified using Image-Pro-Plus software.

To investigate the role of Nrf2, small interfering RNA (siRNA) transfection was utilized to silence Nrf2 expression. Custom-designed Nrf2-siRNA sequences were transfected into chondrocytes using Lipofectamine 2000. Non-targeting siRNA served as a negative control, and successful knockdown was confirmed by qRT-PCR and Western blotting. For in vivo assessment, a mouse model of knee OA was established using medial meniscus surgical destabilization (DMM) in male C57BL/6 mice aged 8 weeks old. Five groups were studied: Sham (arthrotomy only), DMM (OA induction), DMM + low-dose Vin (5 mg kg⁻¹), DMM + high-dose Vin (10 mg kg⁻¹), and DMM + Fer-1 (0.5 mg kg⁻¹). Vin and Fer-1 were administered via intra-articular injections twice weekly for 8 weeks post-surgery [2].

After 8 weeks, mice were euthanized, and knee joints were collected for comprehensive analysis. Radiographic and microcomputed tomography (µCT) examinations were performed to assess the degree of knee joint degeneration. Faxitron X-ray imaging was used for radiographic analysis, while SkyScan-1276 µCT system was employed for 3D structural parameter analysis of knee joints. Bone quality was quantified by measuring bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Osteophyte formation was scored based on number, maturity, and size.

Histopathological analysis involved decalcifying knee joints with 10% ethylenediaminetetraacetic acid (EDTA) for 4 weeks, followed by tissue embedding and sectioning. Safranin O (SO), Toluidine Blue (TB), and hematoxylin-eosin (H&E) staining were performed to evaluate articular cartilage and synovial tissue. Cartilage degeneration was assessed using the International Osteoarthritis Research Society (OARSI) scoring system, and synovitis was evaluated using a validated scoring system. Immunohistochemical staining on mouse knee joint sections that were deparaffinized, hydrated, and antigen-retrieved, were performed using primary antibodies targeting MMP-13, collagen II, GPX-4, and Nrf2, followed by secondary antibody incubation. Quantitative analysis of staining intensity was performed using high-resolution imaging and microscopy [2].

 

Discussion

1) The study by Du et al demonstrated that Vincamine, acting as a GPR40 agonist, significantly improved glucose homeostasis and protected pancreatic β-cell function in both in vitro and in vivo models of type 2 diabetes mellitus (T2DM). These beneficial effects were mediated through complex regulation of the GPR40/cAMP/Ca2+/IRS2/PI3K/Akt signaling pathway. In vitro, Vincamine significantly promoted β-cell survival. In STZ-treated INS-832/13 cells, Vincamine counteracted the STZ-induced decrease in cell viability with an EC50 of 5.78 µM, showing a protective effect against β-cell damage. Flow cytometry analysis confirmed that Vincamine efficiently reduced the mass of apoptotic cells induced by STZ, indicating its anti-apoptotic properties. However, Vincamine itself had no effect on INS-832/13 cell proliferation [1].

Mechanistically, Vincamine protected β-cells by modulating the IRS2/PI3K/Akt signaling pathway. Vincamine effectively reversed the STZ-induced reduction of p-Akt levels with an EC50 of 3.92 µM, as shown by both AlphaLISA and Western blot assays. This protection was dependent on PI3K activity, as wortmannin, a PI3K inhibitor, largely impeded Vincamine’s ability to reverse STZ-induced apoptosis and p-Akt inhibition. Vincamine also recovered the STZ-induced decrease in p-PI3K. Furthermore, Vincamine reversed STZ-induced alterations in downstream effectors: it decreased the STZ-induced increases in Caspase 9/3 activity and cleaved Caspase 3 protein levels, and normalized the depletion of phosphorylated FOXO1 and GSK3β. It also reversed the STZ-induced increases in Bim and p21 mRNA levels. In the upstream pathway, Vincamine antagonized the STZ-induced decrease of IRS2 at both mRNA and protein levels but did not directly affect PI3K enzyme activity or LXRα/β expression. These findings indicate that IRS2 is an upstream component of the PI3K/Akt pathway responding to Vincamine.
Vincamine’s β-cell protection was also strongly linked to cAMP and Ca2+ signaling. Vincamine increased intracellular Ca2+ influx in INS-832/13 cells, which was eliminated by depleting extracellular Ca2+ or by nifedipine, an L-type VDCC inhibitor. Nifedipine also blocked Vincamine’s protective effect against STZ-induced apoptosis and its ability to reverse STZ-induced p-Akt or IRS2 reduction. Vincamine incubation significantly enhanced intracellular cAMP levels, an effect that was inhibited by MDL-12,330A, an adenylyl cyclase inhibitor, or H89, a PKA inhibitor. H89 also hindered Vincamine’s stimulation of p-Akt and IRS2. Intriguingly, Vincamine-mediated Ca2+ influx was found to be cAMP-dependent, as blocking the cAMP pathway eliminated Ca2+ influx changes. This suggests that cAMP and Ca2+ act upstream of the IRS2/PI3K/Akt pathway in Vincamine’s protective effects [1].

Vincamine also promoted glucose-stimulated insulin secretion (GSIS). At a high glucose concentration of 16.8mM, Vincamine effectively promoted GSIS, unlike at low glucose levels of 2.8mM. The GSIS promotion was hindered by MDL-12,330A and KN62, a CaMKII inhibitor, but not by nifedipine or 2-APB, an intracellular Ca2+ release manipulator, suggesting that cAMP and CaMKII pathways are involved in Vincamine-promoted GSIS. It was noted that Vincamine’s β-cell protective effect was not directly related to its GSIS-promoting activity in STZ-treated cells.

A key discovery was that Vincamine functioned as a GPR40 agonist. Using a FLIPR assay in hGPR40-CHO cells, Vincamine was shown to activate GPR40. The Cellular Thermal Shift Assay (CETSA) confirmed direct binding of Vincamine to GPR40, as it stabilized GPR40 against thermal denaturation. Crucially, pharmacological inhibition of GPR40 with GW1100 or GPR40 siRNA transfection blocked Vincamine’s ability to protect against STZ-induced apoptosis and promote p-Akt/IRS2 levels and GSIS. Moreover, GPR40 overexpression in INS-832/13 cells was shown to ameliorate STZ-induced β-cell viability reduction, increase insulin secretion, and enhance cAMP and Ca2+ influx, mimicking the effects of Vincamine itself. This provided strong evidence that Vincamine acts through GPR40 to mediate its protective effects on β-cells and promote GSIS [1].

In vivo experiments further validated Vincamine’s efficacy in animal models of T2DM. In both HFD/STZ and db/db male mice, Vincamine administration of 15 or 30 mg/kg/day, i.p. for 5-6 weeks, significantly improved glucose tolerance. Specifically, it effectively lowered fasting blood glucose levels and HbA1c in both models. Vincamine also ameliorated oral glucose tolerance, as evidenced by reduced blood glucose levels during OGTT, and increased glucose-induced plasma insulin concentration, without affecting basal insulin secretion. Crucially, Vincamine had no effect on insulin sensitivity in vivo as measured by ITT or ex vivo as seen in mouse primary hepatocytes suggesting its primary action is on β-cells rather than peripheral insulin sensitivity [1].

Vincamine also improved pancreatic islet morphology in diabetic mice. In both HFD/STZ and db/db male mice, Vincamine treatment significantly increased the area and number of insulin-positive pancreatic islets, as well as the overall number of β-cells. It also improved the integrity of the islets compared to vehicle-treated groups, without significantly affecting the size of individual β-cells.

The in vivo molecular mechanisms mirrored the in vitro findings, confirming that Vincamine regulates the IRS2/PI3K/Akt signaling pathway in pancreatic islets of diabetic mice. Vincamine administration in HFD/STZ and db/db male mice led to increases in the phosphorylation levels of PI3K, Akt, GSK3β, and FOXO1, along with elevated protein levels of IRS2. Concurrently, Vincamine decreased Caspase 3 enzymatic activity and reduced protein levels of cleaved Caspase 3 in the islets. Immunofluorescence assays confirmed these changes in protein levels of IRS2 and cleaved Caspase 3 within insulin-positive islets, demonstrating that Vincamine’s signaling effects observed in vitro translate directly to the in vivo pancreatic environment [1].

In conclusion, the study provides comprehensive evidence that Vincamine acts as a GPR40 agonist, significantly ameliorating T2DM pathogenesis by improving β-cell function and glucose homeostasis. This is achieved through a multi-targeted mechanism involving the regulation of GPR40/cAMP/Ca2+/IRS2/PI3K/Akt signaling pathways, leading to enhanced β-cell survival and increased glucose-stimulated insulin secretion. The observed improvements in animal models without affecting insulin sensitivity or basal insulin secretion highlight Vincamine’s potential to avoid hypoglycemia, offering a promising therapeutic strategy for T2DM that warrants further investigation [1].

2) The study by Wang et al demonstrated that Vinpocetine (Vin) protects against OA by inhibiting ferroptosis and ECM degradation, primarily through the activation of the Nrf2/GPX4 signaling pathway, as shown in both in vitro and in vivo models. Network pharmacology analysis identified 53 common target genes between Vin and OA, suggesting Vin’s multi-targeted therapeutic potential. GO, KEGG, and Wiki enrichment analyses highlighted Vin’s association with biological processes related to oxidative stress, regulation of ROS metabolism, and cellular components like mitochondria. Ferroptosis, the NRF2 pathway, and arachidonic acid metabolism were identified as crucial pathways. Molecular docking revealed strong binding affinities of Vin for Nrf2 (-5.9 kcal/mol) and GPX4 (-6.7 kcal/mol), with structural modeling indicating hydrogen bonds and other non-covalent interactions within their binding sites [2].

In vitro experiments confirmed Vin’s protective effects against TBHP-induced chondrocyte ferroptosis. TBHP and Erastin significantly increased intracellular ROS levels (DHE and DCFH-DA assays), lipid peroxidation (C11-BODIPY), and Fe2+ accumulation (FerroOrange), while decreasing GPX4 and SLC7A11 expression and increasing ACSL4. Vin (80 and 160 µM) and Fer-1 treatments effectively counteracted these effects, reducing ROS, lipid peroxidation, and Fe2+ levels, and normalizing protein expression of GPX4, SLC7A11, and ACSL4. Vin’s non-cytotoxic profile was confirmed, and it significantly enhanced chondrocyte survival under TBHP stress and notably reduced LDH release, GSH/MDA levels, further validating its anti-ferroptotic action.

Vin also alleviated TBHP-induced mitochondrial dysfunction. TBHP treatment increased mitochondrial ROS, decreased mitochondrial membrane potential (JC-1 assay), and caused mitochondrial fragmentation and morphological abnormalities. Vin at concentrations of 80 and 160 µM, and Fer-1 treatments effectively mitigated these detrimental changes. On a molecular level, Vin restored the expression of mitochondrial dynamics proteins, increasing Opal, Mfn1, and Mfn2 while decreasing Drp1, indicating improved mitochondrial integrity [2].

The activation of the Nrf2/GPX4 pathway by Vin was a central finding. CETSA showed Vin stabilized Nrf2 and GPX4, confirming direct binding. Western blotting demonstrated that Vin promoted Nrf2 release from Keap1, facilitating Nrf2 nuclear translocation and upregulating NQO1, HO-1, and GPX4. Cycloheximide chase assays revealed Vin delayed Nrf2 degradation. Co-immunoprecipitation confirmed Vin decreased Keap1-Nrf2 binding. To validate Nrf2’s role, Nrf2-siRNA knockdown diminished Vin’s protective effects against TBHP-induced ferroptosis and ECM degradation. Nrf2 deficiency reduced Nrf2, NQO1, HO-1, and GPX4 expression, confirming the Nrf2/GPX4 pathway as the key mediator of Vin’s action.

In vivo, a DMM-induced OA mouse model confirmed Vin’s therapeutic efficacy. OA mice exhibited typical symptoms: osteophyte formation, joint space narrowing, and cartilage degradation. Vin, in both low and high doses, and Fer-1 treatments significantly improved these parameters. Radiographic and µCT analyses showed wider joint spaces, smoother joint surfaces, fewer osteophytes, and improved bone quality indicated by BV/TV, Tb.Th, and Tb.N, while reducing Tb.Sp compared to DMM controls. Histopathological staining (SO, TB, H&E) indicated smoother and more intact cartilage surfaces, reduced proteoglycan loss and joint erosion, and significantly lower OARSI scores in Vin-treated groups. Synovitis scores were also substantially reduced. Serum analysis revealed DMM mice had elevated Fe2+ and MDA and reduced GSH, which Vin and Fer-1 treatments effectively normalized. Immunohistochemistry in cartilage tissues demonstrated that DMM increased MMP-13 expression and reduced collagen II, while Vin treatments reversed these changes. Importantly, Vin significantly elevated GPX4 and Nrf2 levels in vivo, corroborating the Nrf2/GPX4 pathway activation [2].

In conclusion, Vin exerts its protective effects in OA by activating the Nrf2/GPX4 signaling pathway, thereby inhibiting ferroptosis and ECM degradation, and mitigating mitochondrial damage. These multi-targeted actions, confirmed by both in vitro and in vivo evidence, position Vin as a promising therapeutic candidate for OA [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Du T, Yang L, Xu X, et al. Vincamine as a GPR40 agonist improves glucose homeostasis in type 2 diabetic mice. J Endocrinol. 2019;240(2):195-214. doi:10.1530/JOE-18-0432

[2] Wang J, Yang J, Fang Y, et al. Vinpocetine protects against osteoarthritis by inhibiting ferroptosis and extracellular matrix degradation via activation of the Nrf2/GPX4 pathway. Phytomedicine. 2024;135:156115. doi:10.1016/j.phymed.2024.156115

 

Vincamine is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.