Main Research Findings

1) Administration of methylene blue has the potential to treat age-related conditions such as neurodegeneration, premature age-related diseases, memory loss, and skin aging.

2) Administration of methylene blue improves hemodynamic stabilization in cases of obstructive jaundice.

 

Selected Data

1) The research team of Xue et examined the potential usage of methylene blue as an anti-aging compound. Methylene blue is a well-known chemical compound that was first synthesized as a textile dye in 1876. Due to its small molecular weight, methylene blue can quickly penetrate tissues, making it effective for various medical applications. Methylene blue can be chemically reduced into leucomethylene blue, a form that possesses strong antioxidant properties. Over the years, methylene blue has gained approval from the U.S. Food and Drug Administration and has been widely utilized in clinical settings, including surgical staining, treatment of malaria, and therapy for methemoglobinemia [1].

The free radicals theory of aging proposes that aging results from accumulated damage caused by free radicals. In human cells, reactive oxygen species, a type of free radical, are predominantly produced in the mitochondria. Aging is associated with a decline in mitochondrial mass, respiration capacity, and efficiency across different tissues. Dysfunctional mitochondria produce less ATP while generating more reactive oxygen species, leading to further mitochondrial damage. This results in significant cellular injury and accelerates the aging process. The antioxidative properties of methylene blue allow it to enhance mitochondrial function and disrupt this damaging cycle. As a result, methylene blue is increasingly recognized as a promising candidate for anti-aging therapies.

The review focuses on the structure, biological functions, and diverse applications of methylene blue. Mitochondria are the primary source of cellular energy in eukaryotic organisms, and the mitochondrial electron transport chain is essential for ATP production. The electron transport chain is located in the inner mitochondrial membrane and involves a series of electron transporters that move electrons from NADH and FADH₂ to molecular oxygen. It is organized into four complexes: the first two complexes NADH-ubiquinone oxidoreductase and succinate dehydrogenase serve as entry points for electrons. Complexes three and four, CoQ-cytochrome c reductase and cytochrome c oxidase further transport the electrons, with the assistance of coenzyme Q10 and cytochrome c. Finally, Complex IV transfers the electrons to molecular oxygen, resulting in the formation of water. During this process, Complex V, ATP synthase, uses the energy generated to phosphorylate ADP into ATP [1].

Under normal physiological conditions, approximately 0.4% to 4% of oxygen is only partially reduced, leading to the generation of reactive oxygen species as a byproduct. Although reactive oxygen species can be produced at multiple sites along the electron transport chain, Complex I is the main source. Mitochondrial dysfunction often impairs Complex I and Complex IV, leading to excessive reactive oxygen species production, oxidative stress, and further cellular damage. In this context, methylene blue plays a critical role in protecting mitochondrial function.

Structurally, methylene blue is a phenothiazine derivative that can exist in both oxidized and reduced forms. The amphipathic nature of methylene blue enables it to easily cross biological membranes. Furthermore, methylene blue is positively charged, which helps it accumulate in mitochondria. Its redox potential is relatively low at 11 millivolts, facilitating its ability to cycle between oxidized and reduced states within mitochondria. This redox cycling ability allows MB to act as a catalytic redox cycler, enhancing the activity of cytochrome oxidase and promoting ATP production [1].

In addition to boosting ATP synthesis, methylene blue can reduce the production of reactive oxygen species by bypassing the dysfunctional activities of Complex I and III. Methylene blue accepts electrons from NADH at Complex I and is reduced to leucomethylene blue, which can then directly donate electrons to cytochrome c, thus being oxidized back to methylene blue. This bypass mechanism helps maintain efficient electron flow even when parts of the electron transport chain are impaired, decreasing reactive oxygen species generation and protecting cells from oxidative stress under pathological conditions [1].

Beyond its mitochondrial effects, methylene blue has been used for various medical purposes. It has long been employed in surgical procedures for staining tissues and in treating conditions like malaria and methemoglobinemia. Early studies from as far back as 1928 demonstrated the ability of methylene blue to photo-inactivate bacteria, and subsequent research revealed potent antiviral properties. Methylene blue has also been found to be effective against fungal and parasitic infections, commonly being used as an aquarium disinfectant even at high concentrations.

Additionally, methylene blue has been explored in photodynamic therapy for several types of cancer, including lung, breast, and prostate cancers. In photodynamic therapy, methylene blue acts as a photosensitizer that, when activated by light, produces cytotoxic species that can kill cancer cells. It is important to note, patients who received methylene blue alongside standard chemotherapy reported no cases of COVID-19 infection among the treated individuals, suggesting a possible protective effect against viral infections [1].

Importantly, studies have shown that low doses of methylene blue, ranging from 0.5 to 4 mg/kg, are effective in stimulating mitochondrial respiration and are safe in both animal models and human subjects. These findings reinforce methylene blue as a safe and versatile drug with broad applications, including potential use as an anti-aging therapy due to its capacity to enhance mitochondrial function, reduce oxidative stress, and interrupt cellular damage pathways associated with aging [1].

2) The research team of Huang et al examine the effects of methylene blue on obstructive jaundice conditions. Obstructive jaundice occurs when the common bile duct is partially or completely blocked, causing bile to leak from the digestive system into the bloodstream, which leads to damage in multiple vital organs. Surgical intervention is critical for relieving this condition; however, such procedures are associated with high rates of postoperative morbidity and mortality. Improved preoperative preparation and perioperative management have been shown to potentially enhance outcomes, but current practices are not yet fully optimized. Patients with obstructive jaundice are particularly vulnerable to severe hypotension after anesthesia induction, often requiring substantial fluid loading and high doses of vasopressors to maintain stable blood pressure [2].

Methylene blue, known as a potent inhibitor of nitric oxide synthase, has been successfully used to counteract refractory hypotension during cardiopulmonary bypass, septic shock, liver transplantation, and anaphylactic shock. While methylene blue is generally considered a second-line therapy for refractory hypotension, evidence suggests that early administration improves hemodynamic outcomes more effectively than delayed use. To examine whether methylene blue has the potential to treat jaundice conditions, researchers enrolled patients scheduled for surgery to relieve obstructive jaundice to evaluate the effects of prophylactic methylene blue administration on hemodynamic stability and organ function.

The study included patients aged 18 to 70 years who exhibited obstructive jaundice, defined by a total bilirubin level greater than 5.0 mg/ml and visible yellowing of the skin or sclera. Exclusion criteria included American Society of Anesthesiologists physical status scores of IV or V, ongoing selective serotonin reuptake inhibitor therapy, a history of abdominal surgery, coronary artery disease, hypertension requiring medication, other cardiovascular conditions, or significant respiratory dysfunction. Patients were randomized in a 1:1 ratio to receive either an intravenous infusion of 2 mg/kg methylene blue diluted in 50 ml saline or 50 ml of saline alone [2].

The methylene blue or placebo solution was administered by continuous intravenous infusion at a rate of 2.5 ml/min before anesthesia induction. Afterward, all patients were induced with 0.02 – 0.05 mg/kg of midazolam, 0.5 – 1 μg/kg of sufentanil, 0.2 – 0.3 mg/kg of etomidate, and 0.2 – 0.3 mg/kg of cisatracurium. Following tracheal intubation, patients were ventilated with a tidal volume of 8–10 ml/kg at 12–16 breaths per minute, maintaining an inspiration-to-expiration ratio of 1:2 and an end-tidal CO₂ level between 35 and 40 cm H₂O. Anesthesia depth was maintained using 1-2% sevoflurane and a continuous intravenous infusion of 2-4 ng/ml of remifentanil, while ensuring a Bispectral Index score between 40 and 60 throughout surgery. Additional boluses of cisatracurium were given as needed based on train-of-four nerve stimulation monitoring.

All patients had central venous and Swan-Ganz catheters placed via the right jugular vein, as well as arterial catheters inserted at the left radial artery site to continuously monitor invasive arterial blood pressure and central venous pressure. Blood gas analyses were conducted at multiple time points including: before and after anesthesia induction, after tumor removal, during entero-anastomosis or choledochojejunostomy, before abdominal closure, and 24 hours postoperatively. Management of hemodynamics and blood transfusion was standardized according to a detailed infusion regimen. Other aspects of intraoperative and postoperative care, such as fluid administration, antibiotics, and pain management, were determined by attending surgeons according to routine clinical practice [2].

The primary outcome measure monitored was the frequency of norepinephrine administration during the intraoperative period. Secondary outcomes included the total dose of norepinephrine, use of other vasoactive agents, volume of fluid administered, liver and renal function parameters, duration of stay in the intensive care unit, and overall hospital stay [2].

 

Discussion

1) Oxidative metabolism is crucial for brain activity, and mitochondrial dysfunction has been linked to neuronal loss during brain aging, as well as to brain diseases such as Alzheimer’s disease, Parkinson’s disease , and brain injuries. The review study conducted by Xue et al reported that methylene blue, due to its lipophilic nature, effectively crosses the blood-brain barrier and accumulates in the brain at higher concentrations than in plasma after administration. Methylene blue has a strong affinity for mitochondria and reduces free radical production by bypassing Complex I/III activity rather than scavenging free radicals directly. Methylene blue can partially restore membrane potential in Complex III-inhibited mitochondria and acts as an electron donor, increasing cytochrome oxidase expression and oxygen consumption. Methylene blue also inhibits nitric oxide, which otherwise suppresses cytochrome c oxidase activity. These properties make methylene blue a promising therapeutic agent for brain diseases [1].

Alzheimer’s disease is a neurodegenerative disease characterized by amyloid-β aggregation and neurofibrillary tangles, and mitochondrial dysfunction may serve as a critical link between aging and Alzheimer’s disease. Elevated oxidative stress from mitochondria is reported early in the progression of Alzheimer’s disease, along with mitochondrial size reduction and impaired movement. Mitochondrial dysfunction in Alzheimer’s disease also leads to impaired energy metabolism, disruptions in oxidative phosphorylation, calcium imbalance, and increased mitochondrial DNA mutations. Low-dose methylene blue treatment can reduce reactive oxygen species production, benefiting Alzheimer’s patients by potentially mitigating mitochondrial damage.

Studies have shown that mitochondrial dysfunction is associated with abnormal amyloid-β and tau processing. Amyloid precursor protein can become trapped in mitochondrial membranes, impairing function, while overexpression of tau also exacerbates mitochondrial damage by reducing ATP and increasing oxidative stress. Conversely, mitochondrial damage can increase abnormal amyloid-β production and tau phosphorylation. Methylene blue has been reported to prevent or dissolve amyloid-β and tau aggregates through autophagic clearance, potentially improving mitochondrial function in neurons related to Alzheimer’s disease. Additionally, methylene blue may directly or indirectly influence β-secretase activity to regulate amyloid-β production. Given that cytochrome oxidase activity declines in Alzheimer’s disease and methylene blue enhances this enzyme’s activity, the nootropic could further support neuronal oxidative metabolism [1].

Clinical studies investigating the efficacy of methylene blue for Alzheimer’s treatment are ongoing. In transgenic mouse models, methylene blue inhibited amyloid-β production and improved cognitive deficits. Low-doses of methylene blue was found to result in an 81% reduction in the rate of cognitive decline over 50 weeks in patients with mild to moderate Alzheimer’s disease, as well as improved cognition and cerebral blood flow. More recent cohort studies suggested a decrease in brain atrophy rates in patients treated with methylene blue, although further research should be conducted [1].

In addition to its effects on the brain, treatment with methylene blue is beneficial for skin aging. The skin, composed of the epidermis, dermis, and subcutis, serves as the body’s primary defense. Skin aging involves elasticity loss, thinning, extracellular matrix degradation, and oxidative stress. Intrinsic aging results from natural processes, while extrinsic aging stems from environmental factors like UV radiation. Oxidative stress plays a role in both types. Methylene blue, as an antioxidant, protects skin by reducing oxidative damage, stimulating cell proliferation, and decreasing aging markers. Studies have shown that treatment with methylene blue increased lifespan and oxygen consumption in fibroblasts, reversed premature senescence, and outperformed traditional antioxidants like vitamin C and retinol. Methylene blue also upregulated collagen and elastin expression, improved skin thickness and hydration, and protected against UV-induced DNA damage.

Administration of methylene blue also facilitates wound healing. Aging reduces fibroblast proliferation and collagen production, impairing skin repair. Methylene blue promotes fibroblast migration and proliferation during wound healing and has been shown to reduce tissue necrosis in rat burn models by decreasing oxidative stress. Additionally, methylene blue has been shown to decrease microbial burden and hyper-granulation while promoting tissue viability with minimal irritation [1].

2) Between January 15, 2017, and June 17, 2018, the research team of Huang et al screened a total of 229 patients for participation in the study investigating methylene blue’s effects during surgery for obstructive jaundice. Of these, 70 patients met the inclusion criteria and were randomly assigned to receive either methylene blue or a saline solution. Blinding of the study participants and investigators was successfully maintained throughout the study. All randomized patients completed the study and were included in the final data analysis. Baseline characteristics between the methylene blue and control groups were well matched, ensuring comparability [2].

The administration of noradrenaline to maintain mean blood pressure above 65 mmHg or greater than 80% of baseline, as well as systemic vascular resistance over 800 dyne/s/cm⁵, was significantly less frequent in the methylene blue group than in the control group. Specifically, 13 out of 35 patients in the methylene blue group required noradrenaline compared to 23 out of 35 in the control group, a difference that was statistically significant. Moreover, the time-averaged dose of noradrenaline administered during the operation was lower in the methylene blue group compared to the control group.

Fewer patients in the methylene blue group required a large total dose of noradrenaline that was defined as more than 2 mg during surgery, with only 1 patient in the methylene blue group versus 9 in the control group. The duration of noradrenaline use was also shorter in the methylene blue group compared to the control group, with median durations of 0 minutes versus 110 minutes, respectively. However, the frequency and dosage of dobutamine use during surgery did not differ significantly between the two groups [2].

At baseline, systolic blood pressure, mean arterial pressure, systemic vascular resistance, and cardiac output were similar between the two groups. Following anesthesia induction, reductions in systolic blood pressure, mean arterial pressure, and systemic vascular resistance were observed in both groups. However, the methylene blue group maintained higher systolic blood pressure and mean arterial pressure values compared to the control group throughout the surgical procedure. Systemic vascular resistance was also significantly higher in the methylene blue group compared to the control group up to 180 minutes after anesthesia induction. Interestingly, cardiac output was higher in the control group 15 minutes after anesthesia induction, and although it remained numerically higher at other time points, these differences were deemed not statistically significant [2].

Plasma transfusion requirements were also lower in the methylene blue group compared to the control group. Specifically, 10 of 35 patients in the methylene blue group received plasma transfusions versus 19 of 35 in the control group. The overall frequency of plasma infusions was also reduced in the methylene blue group by 29% compared to 43%. Despite these differences in plasma transfusion, there were no significant differences between groups in terms of blood loss, use of washed red blood cells, colloid and crystalloid solutions, or other blood products during surgery. Furthermore, the duration of anesthesia and surgery was shorter in the methylene blue group, with anesthesia averaging 7.56 ± 2.13 hours versus 8.79 ± 2.47 hours in the control group and surgical procedures lasting 6.44 ± 2.01 hours versus 7.63 ± 2.31 hours, respectively.

Arterial blood gas analyses showed no significant differences between the two groups in key parameters, including pH, hemoglobin, partial pressures of oxygen and carbon dioxide, potassium, calcium, base excess, and lactic acid levels at all observed time points. In terms of organ function, significantly fewer patients developed acute kidney injury in the methylene blue group compared to the control group by 6% versus 23%, respectively. Additionally, fewer patients in the methylene blue group experienced elevated serum creatinine levels above the normal range on postoperative day 2 by 3% versus 17%, and day 3 by 0% versus 14%, respectively [2].

Regarding liver function, most patients in both groups displayed abnormal liver enzyme levels postoperatively. Elevated glutamic oxaloacetic transaminase levels were observed in 89% of methylene blue patients and 97% of control patients, while elevated glutamic pyruvic transaminase levels occurred in 94% and 97% of patients, respectively, without statistically significant differences. However, glutamic oxaloacetic transaminase levels were significantly lower in the methylene blue group compared to the control group on postoperative days 1, 2, and 3. On day 1, glutamic oxaloacetic transaminase was 255.2 ± 250.3 IU/L in the methylene blue group versus 559.0 ± 837.6 IU/L in the control group. Similar trends were observed on days 2 and 3 [2].

Glutamic pyruvic transaminase levels followed a similar pattern, with significantly lower values in the methylene blue group on days 1 and 2 and a non-significant trend toward lower values on day 3. Finally, creatine kinase activity, an indicator of muscle damage, was also lower in the methylene blue group after surgery. Creatine kinase levels on postoperative day 1 were 441.1 ± 282.3 IU/L in the methylene blue group versus 782.6 ± 480.4 IU/L in the control group, with significant differences persisting on days 2 [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] Xue H, Thaivalappil A, Cao K. The Potentials of Methylene Blue as an Anti-Aging Drug. Cells. 2021 Dec 1;10(12):3379. doi: 10.3390/cells10123379. PMID: 34943887; PMCID: PMC8699482.

[2] Huang, J., Gao, X., Wang, M., Yang, Z., Xiang, L., Li, Y., Yi, B., Gu, J., Wen, J., Lu, K., Zhao, H., Ma, D., Chen, L., & Ning, J. (2023). Prophylactic Administration with Methylene Blue Improves Hemodynamic Stabilization During Obstructive Jaundice-Related Diseases’ Operation: a Blinded Randomized Controlled Trial. Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract, 27(9), 1837–1845.

 

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