Carbon monoxide-loaded red blood cells ameliorate metabolic dysfunction-associated steatohepatitis progression via enhancing AMP-activated protein kinase activity and inhibiting Kupffer cell activation.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive form of nonalcoholic fatty liver disease characterised by fat accumulation, inflammation, oxidative stress, fibrosis, and impaired liver regeneration. In this study, we found that heme oxygenase-1 (HO-1) is induced in both MASH patients and in a MASH mouse model. Further, hepatic carbon monoxide (CO) levels in MASH model mice were >2-fold higher than in healthy mice, suggesting that liver HO-1 is activated as MASH progresses. Based on these findings, we used CO-loaded red blood cells (CO-RBCs) as a CO donor in the liver, and evaluated their therapeutic effect in methionine-choline deficient diet (MCDD)-induced and high-fat-diet (HFD)-induced MASH model mice. Intravenously administered CO-RBCs effectively delivered CO to the MASH liver, where they prevented fat accumulation by promoting fatty acid oxidation via AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor induction. They also markedly suppressed Kupffer cell activation and their corresponding anti-inflammatory and antioxidative stress activities in MASH mice. CO-RBCs also helped to restore liver regeneration in mice with HFD-induced MASH by activating AMPK. We confirmed the underlying mechanisms by performing in vitro experiments in RAW264.7 cells and palmitate-stimulated HepG2 cells. Taken together, CO-RBCs show potential as a promising cellular treatment for MASH.

Carbon monoxide-loaded cell therapy as an exercise mimetic for sarcopenia treatment.

Sarcopenia is characterized by loss of muscle strength and muscle mass with aging. The growing number of sarcopenia patients as a result of the aging population has no viable treatment. Exercise maintains muscle strength and mass by increasing peroxisome growth factor activating receptor γ-conjugating factor-1α (PGC-1α) and Akt signaling in skeletal muscle. The present study focused on the carbon monoxide (CO), endogenous activator of PGC-1α and Akt, and investigated the therapeutic potential of CO-loaded red blood cells (CO-RBCs), which is bioinspired from in vivo CO delivery system, as an exercise mimetic for the treatment of sarcopenia. Treatment of C2C12 myoblasts with the CO-donor increased the protein levels of PGC-1α which enhanced mitochondrial biogenesis and energy production. The CO-donor treatment also activated Akt, indicating that CO promotes muscle synthesis. CO levels were significantly elevated in the skeletal muscle of normal mice after intravenous administration of CO-RBCs. Furthermore, CO-RBCs restored the mRNA expression levels of PGC-1α in the skeletal muscle of two experimental sarcopenia mouse models, denervated (Den) and hindlimb unloading (HU) models. CO-RBCs also restored muscle mass in Den mice by activating Akt signaling and suppressing the muscle atrophy factors myostatin and atrogin-1, and oxidative stress. Treadmill tests further showed that the reduced running distance in HU mice was significantly restored by CO-RBC administration. These findings suggest that CO-RBCs have potential as an exercise mimetic for sarcopenia treatment.

Albumin-fused thioredoxin ameliorates high-fat diet-induced non-alcoholic steatohepatitis.

The pathogenesis of non-alcoholic steatohepatitis (NASH) involves the simultaneous interaction of multiple factors such as lipid accumulation, oxidative stress, and inflammatory response. Here, the effect of human serum albumin (HSA) fused to thioredoxin (Trx) on NASH was investigated. Trx is known to have anti-oxidative, anti-inflammatory, and anti-apoptotic effects. However, Trx is a low molecular weight protein and is rapidly eliminated from the blood. To overcome the low availability of Trx, HSA-Trx fusion protein was produced and evaluated the therapeutic effect on high-fat diet (HFD)-induced NASH model mice. HSA-Trx administered before the formation of NASH pathology showed it to have a preventive effect. Specifically, HSA-Trx was found to prevent the pathological progression to NASH by suppressing lipid accumulation, liver injury markers, and liver fibrosis. When HSA-Trx was administered during the early stage of NASH there was a marked reduction in lipid accumulation, inflammation, and fibrosis in the liver, indicating that HSA-Trx ameliorates NASH pathology. The findings indicate that HSA-Trx influences multiple pathological factors, such as oxidative stress, inflammation, and apoptosis, to elicit a therapeutic benefit. HSA-Trx also inhibited palmitic acid-induced lipotoxicity in HepG2 cells. Taken together, these results indicate that HSA-Trx has potential as a therapeutic agent for NASH pathology.

Carbon Monoxide-Loaded Red Blood Cell Prevents the Onset of Cisplatin-Induced Acute Kidney Injury.

Cisplatin-induced acute kidney injury (AKI) is an important factor that limits the clinical use of this drug for the treatment of malignancies. Oxidative stress and inflammation are considered to be the main causes of not only cisplatin-induced death of cancer cells but also cisplatin-induced AKI. Therefore, developing agents that exert antioxidant and anti-inflammatory effects without weakening the anti-tumor effects of cisplatin is highly desirable. Carbon monoxide (CO) has recently attracted interest due to its antioxidant, anti-inflammatory, and anti-tumor properties. Herein, we report that CO-loaded red blood cell (CO-RBC) exerts renoprotective effects on cisplatin-induced AKI. Cisplatin treatment was found to reduce cell viability in proximal tubular cells via oxidative stress and inflammation. Cisplatin-induced cytotoxicity, however, was suppressed by the CO-RBC treatment. The intraperitoneal administration of cisplatin caused an elevation in the blood urea nitrogen and serum creatinine levels. The administration of CO-RBC significantly suppressed these elevations. Furthermore, the administration of CO-RBC also reduced the deterioration of renal histology and tubular cell injury through its antioxidant and anti-inflammatory effects in cisplatin-induced AKI mice. Thus, our data suggest that CO-RBC has the potential to substantially prevent the onset of cisplatin-induced AKI, which, in turn, may improve the usefulness of cisplatin-based chemotherapy.

A bioinspired carbon monoxide delivery system prevents acute kidney injury and the progression to chronic kidney disease.

Renal ischemia-reperfusion (IR)-induced tissue hypoxia causes impaired energy metabolism and oxidative stress. These conditions lead to tubular cell damage, which is a cause of acute kidney injury (AKI) and AKI to chronic kidney disease (CKD). Three key molecules, i.e., hypoxia-inducible factor-1α (HIF-1α), AMP-activated protein kinase (AMPK), and nuclear factor E2-related factor 2 (Nrf2), have the potential to protect tubular cells from these disorders. Although carbon monoxide (CO) can comprehensively induce these three molecules via the action of mitochondrial reactive oxygen species (mtROS), the issue of whether CO induces these molecules in tubular cells remains unclear. Herein, we report that CO-enriched red blood cells (CO-RBC) cell therapy, the inspiration for which is the in vivo CO delivery system, exerts a renoprotective effect on hypoxia-induced tubular cell damage via the upregulation of the above molecules. Experiments using a mitochondria-specific antioxidant provide evidence to show that CO-driven mtROS partially contributes to the upregulation of the aforementioned molecules in tubular cells. CO-RBC ameliorates the pathological conditions of IR-induced AKI model mice via activation of these molecules. CO-RBC also prevents renal fibrosis via the suppression of epithelial mesenchymal transition and transforming growth factor-ß1 secretion in an IR-induced AKI to CKD model mice. In conclusion, our results confirm that the bioinspired CO delivery system prevents the pathological conditions of both AKI and AKI to CKD via the amelioration of hypoxia inducible tubular cell damage, thereby making it an effective cell therapy for treating the progression to CKD.

Advanced oxidation protein products contribute to chronic kidney disease-induced muscle atrophy by inducing oxidative stress via CD36/NADPH oxidase pathway.

BACKGROUND: Sarcopenia with chronic kidney disease (CKD) progression is associated with life prognosis. Oxidative stress has attracted interest as a trigger for causing CKD-related muscular atrophy. Advanced oxidation protein products (AOPPs), a uraemic toxin, are known to increase oxidative stress. However, the role of AOPPs on CKD-induced muscle atrophy remains unclear. METHODS: In a retrospective case-control clinical study, we evaluated the relationship between serum AOPPs levels and muscle strength in haemodialysis patients with sarcopenia (n = 26, mean age ± SEM: 78.5 ± 1.4 years for male patients; n = 22, mean age ± SEM: 79.1 ± 1.5 for female patients), pre-sarcopenia (n = 12, mean age ± SEM: 73.8 ± 2.0 years for male patients; n = 4, mean age ± SEM: 74.3 ± 4.1 for female patients) or without sarcopenia (n = 12, mean age ± SEM: 71.3 ± 1.6 years for male patients; n = 7, mean age ± SEM: 77.7 ± 1.6 for female ). The molecular mechanism responsible for the AOPPs-induced muscle atrophy was investigated by using 5/6-nephrectomized CKD mice, AOPPs-overloaded mice, and C2C12 mouse myoblast cells. RESULTS: The haemodialysis patients with sarcopenia showed higher serum AOPPs levels as compared with the patients without sarcopenia. The serum AOPPs levels showed a negative correlation with grip strength (P < 0.01 for male patients, P < 0.01 for female patients) and skeletal muscle index (P < 0.01 for male patients). Serum AOPPs levels showed a positive correlation with cysteinylated albumin (Cys-albumin), a marker of oxidative stress (r2  = 0.398, P < 0.01). In the gastrocnemius of CKD mice, muscle AOPPs levels were also increased, and it showed a positive correlation with atrogin-1 (r2  = 0.538, P < 0.01) and myostatin expression (r2  = 0.421, P < 0.05), but a negative correlation with PGC-1α expression (r2  = 0.405, P < 0.05). Using C2C12 cells, AOPPs increased atrogin-1 and myostatin expression through the production of reactive oxygen species via CD36/NADPH oxidase pathway, and decreased myotube formation. AOPPs also induced mitochondrial dysfunction. In the AOPPs-overloaded mice showed that decreasing running time and hanging time accompanied by increasing AOPPs levels and decreasing cross-sectional area in gastrocnemius. CONCLUSIONS: Advanced oxidation protein products contribute to CKD-induced sarcopenia, suggesting that AOPPs or its downstream signalling pathway could be a therapeutic target for the treatment of CKD-induced sarcopenia. Serum AOPPs or Cys-albumin levels could be a new diagnostic marker for sarcopenia in CKD.