Genipin Treatment Research Articles

Genipin ameliorates diabetic retinopathy via the HIF-1α and AGEs-RAGE pathways

BackgroundTraditional Chinese medicine (TCM) is useful in disease treatment and prevention. Genipin is an active TCM compound used to treat diabetic retinopathy (DR). In this study, a network pharmacology (NP)-based approach was employed to investigate the therapeutic mechanisms underlying genipin administration in DR. MethodsThe potential targets of DR were identified using the gene expression omnibus (GEO) database. TCM database screening and NP were used to predict the potential active targets and pathways of genipin in DR. Cell viability was tested in vitro to determine the effects of different doses of glucose and genipin on Human Retinal Microvascular Endothelial Cells (hRMECs). CCK-8, CCK-F, colony formation, CellTiter-Lum, Annexin V-FITC, wound healing, Transwell, tube-forming, reactive oxygen species (ROS), and other assay kits were used to detect the effects of genipin on hRMECs during high levels of glucose. In vivo, a streptozotocin (STZ)-mouse intraocular genipin injection (IOI.) model was used to explore the effects of genipin on diabetes-induced retinal dysfunction. Western blotting was performed to identify the cytokines involved in proliferation, apoptosis, angiogenesis, ROS, and inflammation. The protein expression of the AKT/ PI3K/ HIF-1α and AGEs/ RAGE pathways was also examined. ResultsApproximately 14 types of TCM, and nearly 300 active ingredients, including genipin, were identified. The NP approach successfully identified the HIF-1α and AGEs-RAGE pathways, with the EGR1 and UCP2 genes, as key targets of genipin in DR. In the in vitro and in vivo models, we discovered that high glucose increased cell proliferation, apoptosis, angiogenesis, ROS, and inflammation. However, genipin application regulated cell proliferation and apoptosis, inhibited angiogenesis, and reduced ROS and inflammation in the HRMECs exposed to high glucose. Furthermore, the retinal thickness in the genipin-treated group was lower than that in the untreated group. AKT/ PI3K/ HIF-1α and AGEs/ RAGE signaling was increased by high glucose levels; however, genipin treatment decreased AKT/ PI3K and AGEs/ RAGE pathway expressions. Genipin also increased HIF-1α phosphorylation, oxidative phosphorylation of ATP synthesis, lipid peroxidation, and the upregulation of oxidoreductase. Genipin was found to protect HG-induced hRMECs and the retina of STZ-mice, based on; 1 the inhibition of UCP2 and Glut1 decreased intracellular glucose, and glycosylation; 2 the increased presence of HIF-1α, which increased oxidative phosphorylation and decreased substrate phosphorylation; 3 the increase in oxidative phosphorylation from ATP synthesis increased lipid peroxidation and oxidoreductase activity, and; 4 the parallel effect of phosphorylation and glycosylation on vascular endothelial growth factor (VEGF), MMP9, and Scg3. ConclusionBased on NP, we demonstrated the potential targets and pathways of genipin in the treatment of DR and confirmed its effective molecular mechanism in vitro and in vivo. Genipin protects cells and tissues from high glucose levels by regulating phosphorylation and glycosylation. The activation of the HIF-1α pathway can also be used to treat DR. Our study provides new insights into the key genes and pathways associated with the prognosis and pathogenesis of DR.

Genipin crosslinking promotes biomechanical reinforcement and pro-regenerative macrophage polarization in bioartificial tubular substitutes

Traumatic nerve injuries are nowadays a significant clinical challenge and new substitutes with adequate biological and mechanical properties are in need. In this context, fibrin-agarose hydrogels (FA) have shown the possibility to generate tubular scaffolds with promising results for nerve repair. However, to be clinically viable, these scaffolds need to possess enhanced mechanical properties. In this line, genipin (GP) crosslinking has demonstrated to improve biomechanical properties with good biological properties compared to other crosslinkers. In this study, we evaluated the impact of different GP concentrations (0.05, 0.1 and 0.2% (m/v)) and reaction times (6, 12, 24, 72 h) on bioartificial nerve substitutes (BNS) consisting of nanostructured FA scaffolds. First, crosslinked BNS were studied histologically, ultrastructurally and biomechanically and then, its biocompatibility and immunomodulatory effects were ex vivo assessed with a macrophage cell line. Results showed that GP was able to improve the biomechanical resistance of BNS, which were dependent on both the GP treatment time and concentration without altering the structure. Moreover, biocompatibility analyses on macrophages confirmed high cell viability and a minimal reduction of their metabolic activity by WST-1. In addition, GP-crosslinked BNS effectively directed macrophage polarization from a pro-inflammatory (M1) towards a pro-regenerative (M2) phenotype, which was in line with the cytokines release profile. In conclusion, this study considers time and dose-dependent effects of GP in FA substitutes which exhibited increased biomechanical properties while reducing immunogenicity and promoting pro-regenerative macrophage shift. These tubular substitutes could be useful for nerve application or even other tissue engineering applications such as urethra.

Exogenous Collagen Crosslinking is Highly Detrimental to Articular Cartilage Lubrication.

Healthy articular cartilage is a remarkable bearing material optimized for near-frictionless joint articulation. Because its limited self-repair capacity renders it susceptible to osteoarthritis (OA), approaches to reinforce or rebuild degenerative cartilage are of significant interest. While exogenous collagen crosslinking (CXL) treatments improve cartilage's mechanical properties and increase its resistance to enzymatic degradation, their effects on cartilage lubrication remain less clear. Here, we examined how the collagen crosslinking agents genipin (GP) and glutaraldehyde (GTA) impact cartilage lubrication using the convergent stationary contact area (cSCA) configuration. Unlike classical configurations, the cSCA sustains biofidelic kinetic friction coefficients (μk) via superposition of interstitial and hydrodynamic pressurization (i.e., tribological rehydration). As expected, glutaraldehyde- and genipin-mediated CXL increased cartilage's tensile and compressive moduli. Although net tribological rehydration was retained after CXL, GP or GTA treatment drastically elevated μk. Both healthy and "OA-like" cartilage (generated via enzymatic digestion) sustained remarkably low μk in saline- (≤0.02) and synovial fluid-lubricated contacts (≤0.006). After CXL, μk increased up to 30-fold, reaching values associated with marked chondrocyte death in vitro. These results demonstrate that mechanical properties (i.e., stiffness) are necessary, but not sufficient, metrics of cartilage function. Furthermore, the marked impairment in lubrication suggests that CXL-mediated stiffening is ill-suited to cartilage preservation or joint resurfacing.

Genipin inhibits proliferation of gastric cancer cells by inducing ferroptosis: an integrated study of network pharmacology and bioinformatics study.

Ferroptosis has been demonstrated to suppress cancer development and is targeted for cancer therapy. Genipin, an iridoid constituent in Gardeniae Fructus, has been reported to exert anti-cancer abilities. However, whether genipin could induce ferroptosis remains unclear. The purpose of this study is to explore the anti-gastric cancer (GC) effects of genipin by inducing ferroptosis and to identify the potential targets. CCK-8 and colony formation assays were performed to evaluate the anti-GC effects of genipin. Flowcytometry and western blot were used to indicate ferroptosis-inducing ability of genipin. The potential targets of genipin were analyzed by network pharmacology, screened using UALCAN and KM-plotter database and evaluated by molecular docking. The results showed that genipin inhibited cell viability and proliferation of GC cells. Genipin treatment decreased levels of GPX4 and SLC7A11, induced accumulation of lipid peroxidation intracellularly and led to ferroptosis in GC cells. Network pharmacology analysis identified that lipid- and ROS-related pathways involved in ferroptosis ranked high among genipin-GC common targets. Data from UALCAN and KM-plotter database demonstrated that expression levels of ferroptosis-related targets, including AURKA, BCAT2, DHODH, and GPI, increased in GC tissues and the higher levels of the above four targets were related to tumor stage, tumor grade, and poor prognosis. Among these four targets, AURKA, BCAT2, and DHODH were confirmed by molecular docking with binding energies less than -5. Taken together, our study demonstrates that genipin could exert anti-GC ability by inducing ferroptosis and provides evidence for the potential application of genipin in GC treatment.

Apoptotic activity of genipin in human oral squamous cell carcinoma in vitro by regulating STAT3 signaling.

Genipin, a natural compound derived from the fruit of Gardenia jasminoides Ellis, was reported to have activity against various cancer types. In this study, we determined the underlying mechanism for genipin-induced cell death in human oral squamous cell carcinoma (OSCC). The growth-inhibitory effects of genipin in human OSCC cells was examined by the Cell Counting Kit-8 and soft agar assays. The effects of genipin on apoptosis were assessed by nuclear morphological changes by 4',6-diamidino-2-phenylindole staining, measurement of the sub-G1 population, and Annexin V-fluorescein isothiocyanate/propidium iodide double staining. The underlying mechanism of genipin activity was analyzed by western blot analysis, subcellular fractionation of the nucleus and cytoplasm, immunocytochemistry, and quantitative real-time polymerase chain reaction. Genipin inhibited the growth of OSCC cells and induced apoptosis, which was mediated by a caspase-dependent pathway. Genipin reduced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and its nuclear localization. Furthermore, inhibition of p-STAT3Tyr705 levels following genipin treatment was required for the reduction of survivin and myeloid cell leukemia-1 (Mcl-1) expression, leading to apoptotic cell death. The genipin-mediated reduction in survivin and Mcl-1 expression was caused by transcriptional and/or posttranslational regulatory mechanisms. The results provide insight into the regulatory mechanism by which genipin induces apoptotic cell death through the abrogation of nuclear STAT3 phosphorylation and suggest that genipin may represent a potential therapeutic option for the treatment of human OSCC.

Genipin protects against acute liver injury by abrogating ferroptosis via modification of GPX4 and ALOX15-launched lipid peroxidation in mice.

It is essential to further characterize liver injury aimed at developing novel therapeutic approaches. This study investigated the mechanistic basis of genipin against carbon tetrachloride (CCl4)-triggered acute liver injury concerning ferroptosis, a novel discovered modality of regulated cell death. All experiments were performed using hepatotoxic models upon CCl4 exposure in mice and human hepatocytes in vitro. Immunohistochemistry, immunoblotting, molecular docking, RNA-sequencing and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were conducted. CCl4 intoxication was manifested with lipid peroxidation-dictated ferroptotic cell death, together with changes in a cascade of ferroptosis-associated events and several regulatory pathways. Both the administration of genipin and ferrostatin-1 (Fer-1) significantly prevented this hepatotoxicity in response to CCl4 intoxication via upregulating GPX4 and xCT (i.e., critical regulators of ferroptosis). RNA-sequencing unraveled that arachidonic acid metabolism was considerably influenced upon genipin treatment. Accordingly, genipin treatment attenuated arachidonate 15-lipoxygenase (ALOX15)-launched lipid peroxidation in terms of UHPLC-MS/MS analysis and inflammation. In vitro, genipin supplementation rescued erastin-induced hepatocellular inviability and lipid ROS accumulation. The siRNA knockdown of GPX4 partially abrogated the protective effects of genipin on erastin-induced cytotoxicity, whereas the cytotoxicity was less severe in the presence of diminished ALOX15 expression in L-O2 cells. In conclusion, our findings uncovered that genipin treatment protects against CCl4-triggered acute liver injury by abrogating hepatocyte ferroptosis, wherein the pharmacological modification of dysregulated GPX4 and ALOX15-launched lipid peroxidation was responsible for underlying medicinal effects as molecular basis.

Effects of ultrasound treatment on the morphological characteristics, structures and emulsifying properties of genipin cross-linked myofibrillar protein

Genipin is a natural crosslinker that improves the functional properties of proteins by modifying its structures. This study aimed to investigate the effects of sonication on the emulsifying properties of different genipin concentration-induced myofibrillar protein (MP) cross-linking. The structural characteristics, solubility, emulsifying properties, and rheological properties of genipin-induced MP crosslinking without sonication (Native), sonication before crosslinking (UMP), and sonication after crosslinking (MPU) treatments were determined, and the interaction between genipin and MP were estimated by molecular docking. The results demonstrated that hydrogen bond might be the main forces for genipin binding to the MP, and 0.5 μM/mg genipin was a desirable concentration for protein cross-linking to improve MP emulsion stability. Ultrasound treatment before and after crosslinking were better than Native treatment to improve the emulsifying stability index (ESI) of MP. Among the three treatment groups at the 0.5 μM/mg genipin treatment, the MPU treatment group showed the smallest size, most uniform protein particle distribution, and the highest ESI (59.89%). Additionally, the highest α-helix (41.96%) in the MPU + G5 group may be conducive to the formation of a stable and multilayer oil–water interface. Furthermore, the free groups, solubility, and protein exposure extent of the MPU groups were higher than those of UMP and Native groups. Therefore, this work suggests that the treatment of cross-linking followed by ultrasound (MPU) could be a desirable approach for improving the emulsifying stability of MP.

A Pilot Study to Evaluate Genipin in Staphylococcus aureus and Pseudomonas aeruginosa Keratitis Models: Modulation of Pro-Inflammatory Cytokines and Matrix Metalloproteinases.

Infectious keratitis is a vision-threatening microbial infection. The increasing antimicrobial resistance and the fact that severe cases often evolve into corneal perforation necessitate the development of alternative therapeutics for effective medical management. Genipin, a natural crosslinker, was recently shown to exert antimicrobial effects in an ex vivo model of microbial keratitis, highlighting its potential to serve as a novel treatment for infectious keratitis. This study aimed to evaluate the antimicrobial and anti-inflammatory effects of genipin in an in vivo model of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) keratitis. Clinical scores, confocal microscopy, plate count, and histology were carried out to evaluate the severity of keratitis. To assess the effect of genipin on inflammation, the gene expression of pro- and anti-inflammatory factors, including matrix metalloproteinases (MMPs), were evaluated. Genipin treatment alleviated the severity of bacterial keratitis by reducing bacterial load and repressing neutrophil infiltration. The expression of interleukin 1B (IL1B), interleukin 6 (IL6), interleukin 8 (IL8), interleukin 15 (IL15), tumor necrosis factor-α (TNF-α), and interferon γ (IFNγ), as well as MMP2 and MMP9, were significantly reduced in genipin-treated corneas. Genipin promoted corneal proteolysis and host resistance to S. aureus and P. aeruginosa infection by suppressing inflammatory cell infiltration, regulating inflammatory mediators, and downregulating the expression of MMP2 and MMP9.

Abstract 6121: UCP2 inhibitor eradicates cancer stem-like population in trastuzumab-resistant HER2-positive breast cancer

Abstract Purpose: Uncoupling protein 2 (UCP2) is a member of the mitochondrial anion carrier protein family that plays an important role in stabilizing the inner mitochondrial membrane potential (MMP, ΔΨm) and controlling reactive oxygen species (ROS) production. A selective UCP2 inhibitor, genipin is known to elicit cytotoxicity in several cancers, however, its effects on cancer stem cells (CSCs)-like properties and trastuzumab resistance in HER2-positive breast cancer cells have not been fully elucidated. In the present study, we sought to investigate the mechanism of action of genipin responsible for the induction of apoptosis and its effects on CSC-like features, expression of HER family member and trastuzumab resistance in HER2-positive breast cancer cells in vitro and in vivo. Experimental Designs: The effects of genipin on trastuzuamb-sensitive [BT474 and SKBR3] and trastuzumab-resistant [JIMT-1 and MDA-MB-453] HER2-positive breast cancer cell lines in vitro were evaluated for cell viability, Sub-G1, ROS, MMP, ALDH1 activity, CD44+/CD24- subpopulation and mammosphere formation. To confirm the physiological relevance of our in vitro observations, we explored the impact of genipin on tumor growth and angiogenesis and expression of p95HER2 and ALDH1A1 in trastuzumab-resistant xenograft model in vivo. Results: HER2-positive breast cancer cells harbored a higher level of UCP2, when compared to their counterparts. Genipin significantly downregulated UCP2 and mitochondrial dysfunction coinciding with increased ROS generation and disruption of MMP. These phenomena were accompanied with upregulation of the Bax/Bcl-2 ratio and activation of caspase-3 and caspase-7. Genipin treatment led to significant reduction in levels of truncated p95HER2, p-HER2, p-HER3 and p-Akt levels in both trastuzumab-sensitive and -resistant lines. Marked decline of CD44 and ALDH1A1 expression by genipin treatment was associated with attenuation of mammosphere-forming ability. UCP2 level is predominantly upregulated in CSC-enriched populations, while its knockdown significantly suppressed CSC-like characteristics concomitant with decreased ALDH1A1 and CD44 expression as well as impairment of ALDH1 activity. Genipin administration significantly retarded tumor growth and angiogenesis in trastuzumab-resistant JIMT-1 xenograft tumors. The antitumor effect occurred concomitantly with a decrease in Ki-67 proliferating index and enhancement of apoptosis. Furthermore, individuals receiving genipin exhibited markedly lower levels of p95HER2, full-length p185HER2, CD44 and ALDH1A1 expression compared to their control counterparts. Conclusion: To our knowledge, our findings are the first reported instance of genipin-induced suppression of CSC-like properties and HER2/HER3/Akt axis, implying that genipin treatment may have application in addressing trastuzumab resistance. Citation Format: Minsu Park, Soeun Park, Juyeon Seo, Dongmi Ko, Seongjae Kim, Yong Koo Kang, Kee Dal Nam, So Ra Seuk, Tae-Min Cho, Eunsun Jung, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. UCP2 inhibitor eradicates cancer stem-like population in trastuzumab-resistant HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6121.

Plasma-Polymerised Antibacterial Coating of Ovine Tendon Collagen Type I (OTC) Crosslinked with Genipin (GNP) and Dehydrothermal-Crosslinked (DHT) as a Cutaneous Substitute for Wound Healing

Tissue engineering products have grown in popularity as a therapeutic approach for chronic wounds and burns. However, some drawbacks include additional steps and a lack of antibacterial capacities, both of which need to be addressed to treat wounds effectively. This study aimed to develop an acellular, ready-to-use ovine tendon collagen type I (OTC-I) bioscaffold with an antibacterial coating for the immediate treatment of skin wounds and to prevent infection post-implantation. Two types of crosslinkers, 0.1% genipin (GNP) and dehydrothermal treatment (DHT), were explored to optimise the material strength and biodegradability compared with a non-crosslinked (OTC) control. Carvone plasma polymerisation (ppCar) was conducted to deposit an antibacterial protective coating. Various parameters were performed to investigate the physicochemical properties, mechanical properties, microstructures, biodegradability, thermal stability, surface wettability, antibacterial activity and biocompatibility of the scaffolds on human skin cells between the different crosslinkers, with and without plasma polymerisation. GNP is a better crosslinker than DHT because it demonstrated better physicochemical properties (27.33 ± 5.69% vs. 43 ± 7.64% shrinkage), mechanical properties (0.15 ± 0.15 MPa vs. 0.07 ± 0.08 MPa), swelling (2453 ± 419.2% vs. 1535 ± 392.9%), biodegradation (0.06 ± 0.06 mg/h vs. 0.15 ± 0.16 mg/h), microstructure and biocompatibility. Similarly, its ppCar counterpart, GNPppCar, presents promising results as a biomaterial with enhanced antibacterial properties. Plasma-polymerised carvone on a crosslinked collagen scaffold could also support human skin cell proliferation and viability while preventing infection. Thus, GNPppCar has potential for the rapid treatment of healing wounds.

Degradation and biocompatibility of genipin crosslinked polyelectrolyte films on biomedical magnesium alloy via layer-by-layer assembly

Polyelectrolyte-coated magnesium alloy was prepared by layer-by-layer assembly with poly (allylamine hydrochloride) (PAH) and sodium alginate (ALG). The potential influence of genipin as a crosslinking agent on the microscopic features and corrosion resistance of the coatings was examined using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), potentiodynamic polarization, and electrochemical impedance techniques. An improved anticorrosive property was observed for the crosslinked coating compared to the untreated one, which could be due to the formation of the network barrier against water and corrosion ions such as chloride and oxygen. Furthermore, the cytocompatibility evaluation of different samples by CCK-8 assay suggested that the treatment of genipin was beneficial to the MC3T3-E1 cell proliferation. The study may provide sufficient evidence to show that more efficient LbL assembled multilayer coatings for improving anticorrosion and cytocompatibility would be the ones with crosslinking treatment.

Enhanced gelling properties and hydration capacity of ginkgo seed proteins by genipin cross-linking

The present study investigated the effects of genipin cross-linking on the gelling properties of ginkgo seed protein isolate (GSPI). Cross-linking of GSPI was achieved with different concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.6% w/v) of genipin at pH 6.0. Compared to pure GSPI, genipin treatment led to lower solubility, surface hydrophobicity, and fluorescence intensity, while promoted protein aggregation. Cross-linked GSPI gels exhibited markedly improved gelling properties and water holding capacity (WHC), with up to 2.1-fold increases in gel hardness and 1.3-fold increases in WHC over non-treated GSPI gel. Electrophoresis and Fourier-transform infrared spectroscopy confirmed the cross-linking. Moreover, microstructural examination showed that cross-linking with genipin resulted in protein aggregation and more porous gel matrix. Overall, genipin cross-linking demonstrated great potential for the enhancement of gelling properties of ginkgo seed protein. The current research may expand the utilization of ginkgo seeds in food applications.

Genipin treatment of equine palatal dysfunction: A preliminary study of safety and efficacy

SummaryBackgroundPalatal dysfunction is a common cause of poor performance in racehorses. Although conservative management resolves just over 60% of cases, there is a requirement for further intervention in the residual 40% of cases that do not respond. It is proposed that a palatal stiffening technique that is simple to perform, safe, minimally invasive and enables a rapid return to exercise would be an acceptable first intervention. Genipin is a self‐polymerising molecule that bonds to collagen matrices increasing tissue strength, stiffness and resilience. A previous study demonstrated that implanted genipin increased palatal strength and its resistance to deformation and potentially was effective in decreasing likelihood of palatal dysfunction in horses.ObjectivesThe objective of the study was to appraise the safety and feasibility of implanting genipin oligomers into the equine soft palate and to report on racehorse trainers' subjective assessment of its usefulness in management of palatal dysfunction.Study designProspective, non‐randomised, pilot study.MethodsFifty Thoroughbred racehorses diagnosed with palatal dysfunction were implanted transendoscopically with genipin. The horses were monitored for adverse reactions during hospitalisation. A standard questionnaire provided to the horses' trainers was used to record additional adverse reactions and satisfaction with the animals' subsequent performance. Post‐treatment dynamic endoscopy was performed in a subgroup of animals.ResultsOne horse developed adverse clinical signs of pyrexia and reduced appetite, which responded to treatment. Post‐procedural endoscopic examination revealed mild palatal abnormalities in 6 of 50 horses that showed no clinical signs. One horse had mild generalised oedema, four with mild focal swelling and one with a superficial mucosal ulceration at a single site of implantation of the soft palate. Following treatment, 76% of horses were reported to show clinical improvement by their trainers with 24% reported not to show improvement.Main limitationsThe study was not controlled, and outcome measures were mainly subjective.ConclusionsThe procedure was well tolerated, safe, minimally invasive and enabled a rapid return to exercise. Trainer reported improvement was comparable to previously reported palatoplasty procedures.

Long-Term Study of Corneal Stroma and Endothelium on Structure and Cells After Genipin Treatment of Rabbit Corneas.

PurposeTo study the long-term safety of genipin treatment using a vacuum device with or without epithelial cells at different crosslinking times.MethodsTwenty-five healthy New Zealand white rabbits were separated into five treatment groups: 0.25% genipin with epithelial cells for 5 minutes (G1), 0.25% genipin without epithelial cells for 5 minutes (G2), 0.25% genipin without epithelial cells for 10 minutes (G3), ultraviolet A–riboflavin collagen crosslinking (UVA), and controls (C). Before and 2, 4, 6, and 8 weeks after crosslinking treatment, anterior segment optical coherence tomography (ASOCT), in vivo confocal microscopy (IVCM), and the Pentacam system were used to evaluate the right eyes.ResultsA demarcation line (DL) was observed in the corneal stroma in the G2, G3, and UVA groups. The DL depths in the G2 and G3 groups were stable but decreased in the UVA group over time. The density of keratocytes in these groups increased. Endothelial cell density was decreased in the UVA group. There were no differences in the endothelium before and after treatment in the G1, G2, G3, and C groups. The densitometry, as determined using the Pentacam system, significantly increased in the G2, G3, and UVA groups and was positively correlated with keratocyte densities.ConclusionsA vacuum ring assisting local genipin immersion crosslinking without corneal epithelium can activate the keratocytes in the corneal stroma and was safe enough for the thin cornea.Translational RelevanceGenipin can not only crosslink the collagen fibers but also activate the keratocytes and even may promote collagen fiber secretion.

Genipin Delays Corneal Stromal Enzymatic Digestion.

PurposeTo evaluate the use of genipin in delaying enzymatic digestion of corneal stroma.MethodsHuman corneal stromal tissue was treated with genipin, a known chemical crosslinker, and then along with control tissue was subjected to enzymatic digestion with collagenase. The effects of genipin treatment in retarding stromal digestion were analyzed with phase contrast microscopy, a protein quantification assay, second harmonic generation imaging, and transmission electron microscopy.ResultsGenipin increased stromal resistance to enzymatic digestion when compared with untreated stroma. A morphologic analysis and protein quantification showed increased stromal resistance to enzymatic digestion once stromal tissue was treated with genipin. Second harmonic generation imaging revealed persistent fibrillar collagen signaling in genipin-treated tissue in contrast with untreated tissue suggesting that genipin retards collagenolysis.ConclusionsGenipin increases stromal resistance to enzymatic digestion in controlled experiments as demonstrated by protein quantification studies and through morphologic imaging.Translational RelevanceThis study explores the novel use of genipin in delaying enzymatic stromal digestion. Delaying stromal melting in the setting of corneal infectious or autoimmune keratitis can potentially decrease clinical morbidity.

Effects of genipin crosslinking on mechanical cell-matrix interaction in 3D engineered tendon constructs

It is well known that cells can generate endogenous forces onto the extracellular matrix, but to what extent the mechanical properties of the matrix influences these endogenous cellular forces remains unclear. We therefore sought to quantify the influence of matrix rigidity on cell-matrix interactions by inducing cross-links using increasing concentrations of genipin (0.01–1 mM) or by blocking cross-link formation using beta-aminopropionitrile (BAPN) in engineered human tendon tissue constructs. The cell-matrix mechanics of the tendon constructs were evaluated as cell-generated tissue re-tensioning and stress-relaxation responses using a novel custom-made force monitor, which can apply and detect tensional forces in real-time in addition to mechanical failure testing. Genipin treatment had no influence on the biochemical profile (hydroxyproline, glycosaminoglycan and DNA content) of the constructs and cell viability was comparable between genipin-treated and control constructs, except at the highest genipin concentration. Endogenous re-tension after unloading was significantly decreased with increasing genipin concentrations compared to controls. Mechanical failure testing of tendon constructs showed increased (56%) peak stress at the highest genipin concentration but decreased (72%) with BAPN treatment when compared to controls. Tendon construct stiffness increased with high genipin concentrations (0.1 and 1 mM) and decreased by 70% in BAPN-treated constructs, relative to the controls. These data demonstrate that human tendon fibroblasts regulate their force exertion inversely proportional to increased cross-link capacity but did so independently of matrix stiffness. Overall, these findings support the notion of an interaction between cell force generation and cross-linking, and thus a role for this interplay in mechanical homeostasis of the tissue.

Genipin and insulin combined treatment improves implant osseointegration in type 2 diabetic rats

BackgroundType 2 diabetes mellitus (T2DM) has a harmful effect on the stability and osseointegration of dental implants. T2DM induces mitochondrial damage by inhibiting AMPK signaling, resulting in oxidative stress and poor osteogenesis in the peri-implant bone area. Genipin is a major component of gardenia fruits with strong antioxidant, anti-inflammation, and antidiabetic actions, and it also can activate mitochondrial quality control via the AMPK pathway. The purpose of this study was to investigate the effects of genipin and insulin treatment on implant osseointegration in T2DM rats and explore the underlying mechanisms.MethodsStreptozotocin-induced diabetic rats received implant surgery in their femurs and were then assigned to five groups that were subjected to different treatments for three months: control group, T2DM group, insulin-treated T2DM group (10 IU/kg), genipin-treated T2DM group (50 mg/kg), and the genipin and insulin combination-treated T2DM group. Then, we regularly assessed the weight and glucose levels of the animals. Rats were euthanized at 3 months after the implantation procedure, and the femora were harvested for microscopic computerized tomography analysis, biomechanical tests, and different histomorphometric assessment.ResultsThe results indicated that the highest blood glucose and oxidative stress levels were measured for the T2DM group, resulting in the poorest osseointegration. The combination-treated T2DM group mitigated hyperglycemia and normalized, reactivated AMPK signaling, and alleviated oxidative stress as well as reversed the negative effect of osseointegration. There were beneficial changes observed in the T2DM-genipin and T2DM-insulin groups, but these were less in comparison to the combination treatment group.ConclusionOur study suggests that treatment with genipin in combination with insulin could be an effective method for promoting implant osseointegration in T2DM rats, which may be related to AMPK signaling.

Genipin induces developmental toxicity through oxidative stress and apoptosis in zebrafish

Genipin, an iridoid substance, is mainly derived from Gardenia jasminoides Ellis of the traditional Chinese medicine and is widely used in raw materials for the food additive gardenia blue and biological materials. The developmental toxicity of genipin has not been investigated, and its underlying mechanism is unclear. Therefore, in this study we attempt to investigate the potential developmental toxicity of genipin in zebrafish embryos/larvae. The results showed zebrafish embryos treated with 50 μg/ml dose of genipin display inhibited hatching rates and body length. The pericardial edema was observed. It was also found that genipin could induce cardio-toxicity, hepatotoxicity and nephrotoxicity in zebrafish larvae. After genipin treatment, the suppression of antioxidant capacity and increase of oxidative stress were showed for the triggered generation of ROS and MDA, and decreased activity of SOD. Compared with the 0.5% DMSO group, a number of apoptotic cells in zebrafish were increased after genipin exposure. By measuring marker gene expression with the using of qRT-PCR, we proposed that developmental toxicity after genipin treatment might be associated with oxidative stress and apoptosis increase. Our research offers a better understanding for developmental toxicity of genipin.

Genipin improves reproductive health problems caused by circadian disruption in male mice

BackgroundCircadian rhythm disruption impacts a wide range of physiological processes, including fertility. However, the effect of circadian disruption on male spermatogenesis and fertility, and treatments for these effects have been largely unexplored at the molecular level.MethodsIn this study, we examined the effects of genipin on improving the reproductive health problems caused by circadian disruption. Three groups of animals were fed under different conditions: control group (normal T cycle with saline), group of shortened T cycles (Light/Dark = 4 hours/4 hours) with saline, and a group of shortened T cycles with genipin by oral gavage. The male fertility was evaluated by fertility study and pups parameters analysis after successful sexual behavior and mating with female mice. We sacrificed the treated animals after 5 or 10 weeks and collected the testis, sperm and serum for histological analysis, sperm motility assay, and serum hormone detection, respectively. Furthermore, the effect of genipin was assessed by detection of progesterone secretion and steroidogenic key proteins expression, including StAR and CYP11A1, in mouse Leydig tumor MLTC-1 cells.ResultsMale mice exposed to shortened light-dark cycles, much shorter than 24 hours, had reduced fertility with decreased sperm concentrations and sperm motility. Male mice under circadian disruption have reduced testis size and abnormal morphology, leading to lower fertility rates, reduced litter size and pup body weight. Treatment with exogenous genipin, a natural plant-derived compound, alleviated circadian disruption-induced damage to fertility and spermatogenesis and normalized testosterone, dihydrotestosterone (DHT), and androstenedione (ASD) levels in the male mice. The levels of key proteins involved in steroidogenesis, StAR and CYP11A1, were reduced in mouse testes after the circadian disruption, but genipin treatment restored the reduction. The mRNA expression of SRD5A1, which encodes an androgen synthesis enzyme, was also upregulated by genipin treatment. Furthermore, genipin treatment showed a positive effect on steroidogenesis in MLTC-1 cells, resulting in an increase in hormone secretion and the upregulation of StAR and CYP11A1.ConclusionsOur results showed an association between circadian disruption and reproductive health problems in male mice and indicated that treatments with genipin have positive effects on the reproductive health of male mice with circadian rhythm disorders.

Genipin enhances the antitumor effect of elesclomol in A549 lung cancer cells by blocking uncoupling protein-2 and stimulating reactive oxygen species production.

The uncoupling protein-2 (UCP2) serves a role in tumor aggressiveness and anticancer resistance, which is considered to be associated with its ability to attenuate reactive oxygen species (ROS) production. We hypothesized that UCP2 may protect cancer cells from elesclomol-induced cytotoxicity, and that this may be overcome by blocking UCP2 function with genipin. In A549 lung cancer cells that exhibited high UCP2 expression, treatment with elesclomol alone induced limited changes in glucose uptake, ROS production and cell survival. By contrast, both UCP2 knockdown and genipin treatment mildly reduced glucose uptake, increased ROS production and decreased cell survival. Combining genipin and elesclomol further reduced glucose uptake and increased cellular and mitochondrial ROS production. Moreover, co-treatment with genipin and elesclomol reduced the colony forming capacity to 50.6±7.4% and the cell survival to 42.0±3.4% of that in the control cells (both P<0.001). Suppression of cell survival by treatment with elesclomol and genipin was enhanced in the presence of an exogenous ROS inducer and attenuated by a ROS scavenger. The cytotoxic effects of combining genipin and elesclomol were accompanied by reduced mitochondrial membrane potential and occurred through apoptosis as demonstrated by Annexin V assay and increased protein cleavage of PARP and caspase-3. Finally, in an A549 ×enograft mouse model, tumor growth was only modestly retarded by treatment with elesclomol or genipin alone, but was markedly suppressed by combining the two drugs compared with that in the control group (P=0.008). Therefore, high UCP2 expression may limit the antitumor effect of elesclomol by attenuating ROS responses, and this may be overcome by co-treatment with genipin; combining elesclomol and genipin may be an effective strategy for treating cancers with high UCP2.