Corm Research Articles

Development of Manganese Carbonyl Loaded Upconversion Nanoparticles for Near‐Infrared‐Triggered Carbon Monoxide and Mn2+ Delivery

Photoactivatable carbon monoxide‐releasing molecules (CORMs), typically based on transition‐metal carbonyl complexes, have reliance on activation by UV or visible light that restricts their biomedical applications. To address this limitation, a near‐infrared (NIR)‐responsive nanoplatform is presented based on upconversion nanoparticles (UCNPs) loading with manganese carbonyl complex Mn2(CO)10 that concurrently releases CO and manganese ion (Mn2+). With the UCNPs, the more tissue‐penetrable NIR is used to locally generate UV light for photodecomposition of Mn2(CO)10 into CO and manganese oxide (MnOX), after which MnOX is reduced to Mn2+ by the overexpressed glutathione in cancer cells. Moreover, the released Mn2+ can serve as a magnetic resonance imaging contrast agent to monitor the NIR‐controlled corelease of CO and Mn2+ in real time. Therefore, this nanoplatform can provide a potential strategy for NIR‐enabled spatiotemporally release of CO and Mn2+, enhancing the controlled delivery and biomedical application of CORMs.

An orally active carbon monoxide-releasing molecule enhances beneficial gut microbial species to combat obesity in mice

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, β-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction in inflammatory and metabolic profiles. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

Excited-State Intramolecular Proton Transfer-Driven Photon-Gating for Photoelectrochemical Sensing of CO-Releasing Molecule-3.

Different from prevalent approaches such as immunological recognition, complementary base pairing, or enzymatic regulation in current photoelectrochemical (PEC) sensing, this study reported an excited-state intramolecular proton transfer (ESIPT)-driven photon-gating PEC sensor. The sensor is developed for the detection of CO-releasing molecule-3 (CORM-3) by modifying an ESIPT-switched organic fluorescent probe molecule (NDAA) onto the surface of a p-type semiconductor (BiOI). The NDAA can be excited and exhibit strong green fluorescence after responding with CORM-3, resulting in an electrode-interface photon competitive absorption effect due to the switch on ESIPT and considerably reducing the photocurrent signal. The experimental results revealed that the as-developed PEC sensor achieved good analytical performance with high selectivity and sensitivity, with a linear range of 0.01-1000 μM and a lower detection limit of 6.5 nM. This work demonstrates the great potential of the organic fluorescent probe molecule family in advancing PEC analysis. It is anticipated that our findings will stimulate the creation of diverse functional probes possessing distinctive characteristics for inventive PEC sensors.

Azoanthracene-core structure as Cu2+-assisted CO sensing probe: Characterization, performance, and bioimaging

Detection of endogenous CO (carbon monoxide) is an interesting topic in biology because it has been discovered as a messenger for signal transduction and therapeutic effects in vital biological activities. Fluorescence imaging has proven a powerful tool for detecting endogenous CO, which drives the development of low-cost and easy-to-use fluorescent probes. In this study, four azobenzene derivatives (A1, A2, A3, and A4) with various substituents were reported, including their geometric structures, photophysical parameters, and spectral responses to Cu2+ and CO. The relationship between substituent structure and performance was discussed along with Cu2+ quenching and CO sensing mechanisms. The optimal probe (A1), which had no substituent, efficiently quenched fluorescence in the presence of Cu2+, with its PLQY decreased from 0.33 to 0.02, PLQY = photoluminescence quantum yield. Upon CO deoxidization, A1′s fluorescence could be recovered (PLQY recovered to 0.32) within 180 s. Its sensing mechanism was static by forming a non-fluorescent complex with Cu2+ (with a stoichiometric ratio of 1:1). The bioimaging performance of A1 for endogenous CO in HeLa cells was reported.

Neutral and Cationic Re(I) Complexes with Pnictogen-Based Coligands and Tunable Functionality: From Phosphorescence to Photoinduced CO Release.

In this work, we have explored Re(I) complexes featuring triphenylpnictogen (PnPh3, Pn = P, As, or Sb)-based coligands and bidentate (neutral or monoanionic) luminophores derived from 1,10-phenantroline (phen), as well as from 2-(3-(tert-butyl)-1H-1,2,4-triazol-5-yl)pyridine (H(N-tBu)). The effect of the increasingly heavy elements on the structural parameters, photoexcited-state properties, and electrochemical behavior as well as the hybridization defects and polarization of the Pn atoms was related to the charges of the main luminophores (i.e., phen vs N-tBu) and explored in terms of photoluminescence spectroscopy, X-ray diffractometry, and quantum-chemical methods. Therefore, an in-depth analysis of the bonding, crystal packing, excited-state energies, and lifetimes was assessed in liquid solutions, frozen glassy matrices, and crystalline phases along with a semiquantitative photoactivation study. Notably, by changing the main ligand from phen to N-tBu, an increase in radiative and radiationless deactivation rates (kr and knr, respectively) at 77 K together with a faster photoinduced CO release and fragmentation at room temperature was detected. In addition, a progressively red-shifted phosphorescence was observed with the growing atomic number of the pnictogen atom, along with a boost in kr and knr at 77 K. Down the Vth main group and upon coordination of the Pn atom to the Re(I) center, an increasingly prominent jump of s-orbital participation on the binding sxp3.00-orbitals of the Pn atoms is evidenced. Based on these findings, the ability of these complexes to act as tunable photoluminescent labels able to perform as light-driven CO-releasing molecules is envisioned.

Exogenous carbon monoxide promotes GPX4-dependent ferroptosis through ROS/GSK3β axis in non-small cell lung cancer

The gas therapy is drawing increasing attention in the treatment of many diseases including cancer. As one of gas signaling molecules, carbon monoxide (CO) has been proved to exert anti-cancer effects via triggering multiple cell death types, such as autophagy, apoptosis and necrosis. Here, we showed that low concentration CO delivered from CO-releasing molecule 3 (CORM-3) effectively induced ferroptosis, known as a novel proinflammatory programmed cell death, in vitro and in vivo. Mechanistically, we found that CO triggered ferroptosis by modulating the ROS/GSK3β/GPX4 signaling pathway, resulting in the accumulation of lipid hydroperoxides and the occurrence of ferroptosis. We think our findings provide novel insights into the anti-cancer mechanisms of CO, and suggest that CO could potentially be exploited as a novel ferroptosis inducer for cancer treatment in the future.

Cell membrane-targeted surface enhanced Raman scattering nanoprobes for the monitoring of hydrogen sulfide secreted from living cells

Hydrogen sulfide (H2S), an important gas signal molecule, participates in intercellular signal transmission and plays a considerable role in physiology and pathology. However, in-situ monitoring of H2S level during the processes of material transport between cells remains considerably challenging. Herein, a cell membrane-targeted surface-enhanced Raman scattering (SERS) nanoprobe was designed to quantitatively detect H2S secreted from living cells. The nanoprobes were fabricated by assembling cholesterol-functionalized DNA strands and dithiobis(phenylazide) (DTBPA) molecules on core-shell gold nanostars embedded with 4-mercaptoacetonitrile (4-MBN) (AuNPs@4-MBN@Au). Thus, three functions including cell-membrane targeted via cholesterol, internal standard calibration, and responsiveness to H2S through reduction of azide group in DTBPA molecules were integrated into the nanoprobes. In addition, the nanoprobes can quickly respond to H2S within 90 s and sensitively, selectively, and reliably detect H2S with a limit of detection as low as 37 nM due to internal standard-assisted calibration and reaction specificity. Moreover, the nanoprobes can effectively target on cell membrane and realize SERS visualization of dynamic H2S released from HeLa cells. By employing the proposed approach, an intriguing phenomenon was observed: the other two major endogenous gas transmitters, carbon monoxide (CO) and nitric oxide (NO), exhibited opposite effect on H2S production in living cells stimulated by related gas release molecules. In particular, the introduction of CO inhibited the generation of H2S in HeLa cells, while NO promoted its output. Thus, the nanoprobes can provide a robust method for investigating H2S-related extracellular metabolism and intercellular signaling transmission.

Synthesis, structural characterization, docking studies and bioactivities of carbon monoxide release molecules based on triphenylphosphonium-glycyrrhetinic acid derivatives

Carbon monoxide-releasing molecules (CORMs) containing cobalt complexes based on triphenylphosphonium-glycyrrhetinic acid (GA-TPP) were synthesized. This includes cytotoxicity, mitochondrial apoptosis, molecular docking, as well as western blotting.

Near-infrared fluorogenic imaging of carbon monoxide in live cells using palladium-mediated carbonylation.

Here we develop a near infrared (NIR) fluorogenic probe for carbon monoxide (CO) detection and imaging based on palladium-mediated carbonylation using a NIR boron-dipyrromethene difluoride as a fluorophore and tetraethylene glycols as aqueous moieties. The probe is utilized to image exogenous and endogenous CO under different stimulated conditions in live cells.

A long-wavelength mitochondria-targeted CO fluorescent probe for living cells and zebrafish imaging.

Carbon monoxide (CO) not only causes damage to life and health as an environmental pollutant, but also undertakes many physiological functions in organisms. In particular, developing means that can be used for the determination of CO in organelles will provide insight into the vital role it plays. Studies have shown that mitochondrial respiration is closely related to CO concentrations, so it is critical to develop tools for CO detection in mitochondria. Here, we use a rhodamine derivative that can target mitochondria as fluorophores to construct a mitochondrial-labeled CO fluorescence probe (Rh-CO) with high sensitivity (detection limit: 9.4 nM), excellent water-solubility, and long emission (λem = 630 nm). Prominently, the probe has outstanding mitochondria-targeting capabilities. Moreover, we used transient glucose deprivation (TGD) and heme to stimulate endogenous CO production in living cells and zebrafish, respectively, and the probe exhibited excellent imaging capabilities. All in all, we expect this probe to contribute to a deeper understanding of the role played by CO in mitochondria.

Lysosome targeted visible light-induced photo-CORM for simultaneous CO-release and singlet oxygen generation.

We report a specific lysosome targeted light-responsive CO-releasing molecule (Lyso-CORM). Lyso-CORM is very stable under dark conditions. CO and singlet oxygen (1O2) generation was effectively triggered under one photon and two photon excitation (800 nm) conditions. The cytotoxicity results demonstrated that Lyso-CORM showed good phototoxicity due to the synergistic effect of CO and 1O2 release, and its good biocompatibility, negligible dark toxicity and specific lysosome targeting make Lyso-CORM a potent candidate for phototherapeutic applications.

Photoinduced CO-releasing molecule (photoCORM) as an in situ CO surrogate for palladium-catalysed aminocarbonylation.

Herein, we show that a photoCORM behaves as a highly substrate-tolerant, photoactivable in situ CO surrogate, allowing the synthesis of variously functionalised amides including the biologically active compound Moclobemide by Pd-catalysed aminocarbonylation between aryl iodides and amines under mild conditions. This work should bring about a useful input to the field of carbonylative transformations.

Endogenous CO imaging in bacterial pneumonia with a NIR fluorescent probe

Bacterial pneumonia is a serious respiratory illness that poses a great threat to human life. Rapid and precise diagnosis of bacterial pneumonia is crucial for symptomatic clinical treatment. Endogenous carbon monoxide (CO) is regarded as a significant indicator of bacterial pneumonia; herein, we developed a near-infrared (NIR) probe for fluorescence and photoacoustic (PA) dual-mode imaging of endogenous CO in bacterial pneumonia. NO2-BODIPY could rapidly and specifically react with CO to produce strong NIR fluorescence as well as ratiometric PA signals. NO2-BODIPY has outstanding features including fast response, fluorescence/PA dual mode signals, good specificity, and a low limit of detection (LOD = 20.3 nM), which enables it to image endogenous CO in cells and bacterial pneumonia mice with high sensitivity and high contrast ratio. In particular, NO2-BODIPY has two-photon excited (1340 nm, σ1 = 1671 GM) NIR fluorescence and has been utilized to image endogenous CO in bacterial pneumonia mice with deep tissue penetration. NO2-BODIPY has been demonstrated a good capability of fluorescence/PA dual-mode imaging of CO in bacterial pneumonia mice, providing a precise manner to diagnose bacterial pneumonia.

Simultaneous Light-Triggered Release of Nitric Oxide and Carbon Monoxide from a Lipid-Coated Upconversion Nanosystem Inhibits Colon Tumor Growth.

Gas therapy has gained noteworthy attention in biomedical research, with the rise of gas-releasing molecules enhancing their therapeutic potential, especially when integrated into nano-based drug delivery systems. Herein, we present a lipid-coated gas delivery system to simultaneously shuttle two gas-releasing molecules carrying nitric oxide (NO) and carbon monoxide (CO), respectively. Upconversion nanoparticles (UCNPs) are designed to generate photons at 360 nm upon 808 nm of near-infrared (NIR) irradiation. These in situ-generated UV photons trigger simultaneous NO and CO release from S-nitrosoglutathione (GSNO) and the CO-releasing molecule (CORM), respectively, which are coloaded into lipid-coated UCNP/GSNO/CORM/FA nanoparticles (LUGCF). LUGCF with a GSNO/CORM mass ratio of 2:1 is determined to be optimal in terms of synergistically instigating apoptosis in HCT116 and CT26 colon cancer cells, where both NO/CO are released and subsequent production of ROS are detected. This CO/NO combination nanoplatform exhibits a very effective inhibition of colon tumor growth in vivo at relatively low doses upon a mild 808 nm irradiation. Overall, we effectively integrated two therapeutic gas-releasing molecules in one NIR-responsive nanosystem, presenting a promising therapeutic strategy for future biomedical applications in dual-gas cancer therapy.

The Role of Red Blood Cell Lifespan Testing in the Evaluation of Patients with Paroxysmal Nocturnal Hemoglobinuria

Objective To explore the role of red blood cell(RBC) lifespan testing in the evaluation of patients with paroxysmal nocturnal hemoglobinuria (PNH). Methods The endogenous breath carbon monoxide (CO) test was used to detect the RBC lifespan of PNH patient with the treatment of crovalimab（ Roche） or iptacopan（ Novartis.），for comparison of different hemolysis indices in the assessment of patients' condition. Results Nine patients have been enrolled in the study at our centre from June to December 2021, 4 with the treatment of crovalimab and 5 with iptacopan. The median lifespan in the untreated patients was 17 (range 12-38) days, median hemoglobin(Hb) 83(69-94) g/l, median lactic dehydrogenase（LDH）590.8(446.1-935.5)U/L, median PNH clones 96.0(33.2-99.9)%. By 6 months post-treatment, 8 patients achieved hematologic improvement without breakthrough hemolysis. In all patients, LDH levels decreased rapidly and stabilized at lower levels after starting treatment. But the degree of Hb recovery varied，median 60(20-85)g/L. Analyzing the correlation between different hemolytic indices (LDH, RBC lifespan, total bilirubin, indirect bilirubin, etc.) and Hb changes, RBC lifespan showed good correlation（P=0.002，R=0.867）. In the crovalimab group, three patients presented with a positive Coombs test and the negative patient didn't obtain hematologic improvement. No patients with a positive Coombs test were seen in the iptacopan group. Conclusion Different (upstream/downstream) complement inhibitors have different effects on the condition of PNH patients, and different indicators can reflect different results; LDH responds to intravascular hemolysis, and red blood cell lifespan also quantifies extravascular hemolysis, and is applied to monitor hemolysis. The application of RBC lifespan testing can provide an accurate scientific basis for the assessment of PNH conditions. Keywords: red blood cell lifespa, paroxysmal nocturnal hemoglobinuria ,red blood cell lifespan,lactic dehydrogenase

Relationship between red blood cell lifespan and endogenous carbon monoxide in the common bottlenose dolphin and beluga.

Certain deep-diving marine mammals [i.e., northern elephant seal (Mirounga angustirostris), Weddell seal (Leptonychotes weddellii)] have blood carbon monoxide (CO) levels that are comparable with those of chronic cigarette smokers. Most CO produced in humans is a byproduct of heme degradation, which is released when red blood cells (RBCs) are destroyed. Elevated CO can occur in humans when RBC lifespan decreases. The contribution of RBC turnover to CO concentrations in marine mammals is unknown. Here, we report the first RBC lifespans in two healthy marine mammal species with different diving capacities and heme stores, the shallow-diving bottlenose dolphin (Tursiops truncatus) and deep-diving beluga whale (Delphinapterus leucas), and we relate the lifespans to the levels of CO in blood and breath. The belugas, with high blood heme stores, had the longest mean RBC lifespan compared with humans and bottlenose dolphins. Both cetacean species were found to have three times higher blood CO content compared with humans. The estimated CO production rate from heme degradation indicates some marine mammals may have additional mechanisms for CO production, or delay CO removal from the body, potentially from long-duration breath-holds.NEW & NOTEWORTHY This is the first study to determine the red blood cell lifespan in a marine mammal species. High concentrations of carbon monoxide (CO) were found in the blood of bottlenose dolphins and in the blood and breath of belugas compared with healthy humans. Red blood cell turnover accounted for these high levels in bottlenose dolphins, but there may be alternative mechanisms of endogenous CO production that are contributing to the CO concentrations observed in belugas.

A novel fluorescent probe for ultra-fast detection of CORM-3 and spatiotemporal multi-organelle targeting

Endogenous CO is produced by heme in cells and is involved in various physiological processes. Carbon monoxide releasing molecules (CORMs) could deliver CO-controlled to biological systems. At present, most fluorescent probes used to detect CO or CORM have some problems, such as long response time and toxicity to biological systems. Therefore, this work reports a novel fluorescent probe (PNQ) that can detect CORM-3 real-time based on the coordination of the intramolecular electron-poor double bond and the reduction of nitro group by green and red fluorescence channels. A detail recognition mechanism is proposed. The probe enables ultra-fast detection of CORM-3 without the assistance of other substances and monitors CORM-3 concentrations at the cellular level. Significantly, PNQ is the first case that can detect the concentration of CORM-3 in endoplasmic reticulum and mitochondria based on different fluorescence emission and has the characteristics of spatial and temporal distribution. We believe that this probe has great potential for studying synergistic or antagonistic effects between organelles on CO released by CORM-3 and regulation.

Exploring the therapeutic potential of a nano micelle containing a carbon monoxide-releasing molecule for metabolic-associated fatty liver disease by modulating hypoxia-inducible factor-1α.

Metabolic-associated fatty liver disease (MAFLD) encompasses a spectrum of chronic liver diseases, including steatohepatitis, cirrhosis, and liver cancer. Despite the increasing prevalence and severity of MAFLD, no approved pharmacological interventions are currently available. Hypoxia-inducible factor-1α (HIF-1α) has emerged as a crucial early mediator in the pathogenesis of MAFLD. Previously, we demonstrated the potent anti-inflammatory properties of the nano-designed carbon monoxide (CO) donor, styrene maleic acid copolymer (SMA) encapsulating CO-releasing molecule (SMA/CORM2), which effectively suppressed HIF-1α in various inflammatory disorders. Here, we investigated the therapeutic potential of SMA/CORM2 in a mouse model of MAFLD induced by a high-fat methionine- and choline-deficient (HF-MCD) diet. Following 4 weeks of HF-MCD diet consumption, we observed pronounced hepatic lipid accumulation accompanied by disrupted lipid metabolism, polarization of macrophages towards the pro-inflammatory M1 phenotype, activation of the NLRP3 inflammasome, and upregulation of the TGF-β fibrosis signaling pathway. Notably, the early and upstream event driving these pathological changes was the upregulation of HIF-1α. Treatment with SMA/CORM2 (10mg/kg, three times per week) led to a significant increase in CO levels in both the circulation and liver, resulting in remarkable suppression of HIF-1α expression even before the onset of apparent pathological changes induced by the HF-MCD diet. Consequently, SMA/CORM2 administration exerted a significantly protective and therapeutic effect on MAFLD. In vitro studies using hepatocytes treated with high concentrations of fatty acids further supported these findings, as knockdown of HIF-1α using short hairpin RNA (shRNA) elicited similar effects to SMA/CORM2 treatment. Collectively, our results highlight the therapeutic potential of SMA/CORM2 in the management of MAFLD through suppression of HIF-1α. We anticipate that SMA/CORM2, with its ability to modulate HIF-1α expression, may hold promise for future applications in the treatment of MAFLD. STATEMENT OF SIGNIFICANCE: Carbon monoxide (CO) is a crucial gaseous signaling molecule that plays a vital role in maintaining homeostasis and is a potential target for treating many inflammatory diseases. Developing drug delivery systems that can deliver CO stably and target specific tissues is of great interest. Our team previously developed a nano micellar CO donor, SMA/CORM2, which exhibits superior bioavailability to native CORM2 and shows therapeutic potential in many inflammatory disease models. In this study, we showed that SMA/CORM2, through controlled CO release, significantly ameliorated steatohepatitis and liver fibrosis induced by an HF-MCD diet by suppressing an HIF-1α mediated inflammatory cascade. These findings provide new insight into the anti-inflammatory function of CO and a promising approach for controlling metabolic-associated fatty liver disease.

Polysiloxane probes with main chain modifications regulating subcellular organelles localization for dynamic observation of lysosomal and mitochondrial interactions during cellular hypoxia

As an important pathological state, hypoxia is closely associated with the occurrence of numerous diseases. The degree of hypoxia can reflect the related pathological conditions. The expression of heme oxygenase-1 (HO-1) will lead to the increase of endogenous carbon monoxide (CO) during hypoxia. Abnormal concentrations of CO involved in regulating various physiological activities of cells may result in the damage of organelles from lysosomes to mitochondria and further disrupt the normal communication between organelles. However, normal intercellular communication and interactions have profound significance for the dynamic equilibrium of the intracellular environment. Hence, it is a challenging task to develop new tools to visualize the effect of CO on the interaction of different organelles during hypoxia. In this paper, we designed fluorescent probes that could switch between lysosomes and mitochondria by using polysiloxane platforms (P1-CORM-Lyso and P1-CORM-Mito) for the first time. P1-CORM-Lyso and P1-CORM-Mito successfully enabled monitor the CO generated during anoxia with excellent biocompatibility. We expect that this switchable targeting silicon-based fluorescent materials will open up new fields for the design of new fluorescent probes and get a deeper understanding of the cellular hypoxia process.

The effects of CORM3 or NaHS on the oxidative stress caused by chronic kidney disease in rats: potential interaction between CO and H2S signaling pathway.

Neurotoxicity is implicated as a severe complication of chronic kidney disease (CKD). Accumulation of urea and other toxic compounds leads to oxidative stress, inflammation and destruction of the blood-brain barrier. Carbon monoxide (CO) and hydrogen sulfide (H2S) have been shown to have anti-inflammatory, anti-apoptotic, and anti-proliferative properties. The aims of the present study were evaluated the protective effects of CO-releasing molecule (CORM3) and H2S donor (NaHS) on oxidative stress and neuronal death induced by CKD in the hippocampus and prefrontal cortex by considering interaction between CO and H2S on CBS expression. CORM3 or NaHS significantly compensated deficits in the antioxidant defense mechanisms, suppressed lipid peroxidation and reduced neuronal death in hippocampus and prefrontal cortex and improvement the markers of renal injury that induced by CKD. In addition, CORM3 or NaHS significantly improved CBS expression which were reduced by CKD. However, improving effects of CORM3 on antioxidant defense mechanisms, lipid peroxidation, neuronal death, renal injury and CBS expression were prevented by amino-oxy acetic acid (AOAA) (CBS inhibitor) and reciprocally improving effects of NaHS on all above indices were prevented by zinc protoporphyrin IX (Znpp) (HO-1 inhibitor). In conclusion, this study demonstrated that formation of CO and H2S were interdependently improved CKD-induced oxidative stress and neuronal death, which is may be through increased expression of CBS.