SIRT3 ( IC50 = 16 nM ); SIRT1 ( IC50 = 88 nM ); SIRT2 ( IC50 = 92 nM )

3-TYP (50 mg/kg, i.p.) has no discernible impact on the LVEF, LVFS, infarct size, serum LDH levels, oxidative stress, or apoptosis when compared to the Sham group's values. Furthermore, when compared to the Sham group, 3-TYP has minimal impact on the expression levels of gp91phox, Nrf2, NQO 1, Bax, Bcl-2, Caspase-3, and cleaved Caspase-3. 3-TYP has no effect on SIRT3 expression, but it dramatically reduces SIRT3 activity and increases SOD2 acetylation in comparison to the control group. 3-TYP lowers the LVEF and LVFS following a 24-hour period of reperfusion, which lessens the cardioprotective effects of melatonin. In addition, compared to individuals in the IR+Mel group, 3-TYP increases the apoptotic ratio, serum LDH levels, and infarct size[2].

SIRT3 inhibitor (3-TYP) was used to confirm that melatonin was involved in Cd-induced autophagy and the disruption of SIRT3-regulated mitochondrial-derived O2•− production. 3-TYP is a selective SIRT3 inhibitor.31 Exposure to 3-TYP inhibited melatonin-enhanced SIRT3 activity but did not affect SIRT3 protein expression (Fig. S6A and B). Moreover, 3-TYP pretreatment reversed the protective effects of melatonin on Cd-induced mitochondrial-derived O2•− production and autophagic cell death (Fig. 9A–C and Fig. S3F). As shown in Figures 9D and 9E, melatonin-induced increases in deacetylated-SOD2 expression and SOD2 activity were significantly attenuated by 3-TYP in HepG2 cells exposed to Cd[1].

In brief, a 6-0 silk suture slipknot is wrapped around the left anterior descending coronary artery to temporarily exteriorize the heart in male C57BL/6 mice under 2% isoflurane anesthesia. Following 30 minutes of myocardial ischemia, the myocardium is reperfused for 3 hours (to measure oxidative stress and perform a western blot analysis) or 24 hours (to assess infarct size, cardiac function, and apoptotic index). The slipknot is then released. The identical surgical procedures are performed on sham-operated mice, with the exception that the suture under the left coronary artery is left untied. Mice are randomized to receive an intraperitoneal injection of either melatonin (20 mg/kg) or vehicle (1% ethanol) ten minutes prior to reperfusion. The C57BL/6 mice are split into the following groups at random: (i) Sham group: mice underwent the sham operation and are treated with vehicle (1% ethanol); (ii) Mel group: mice are treated with melatonin (20 mg/kg via intraperitoneal injection); (iii) IR+V group: mice underwent the MI/R operation and are treated with vehicle (1% ethanol); (iv) IR+Mel group: mice underwent the MI/R operation and are treated with melatonin (20 mg/kg via intraperitoneal injection 10 minutes before reperfusion); (v) IR+Mel+3-TYP group: mice are pretreated with 3-TYP (3-TYP is intraperitoneally injected at a dose of 50 mg/kg every 2 days for a total of three doses prior to the MI/R surgery), subjected to the MI/R operation, and treated with melatonin (20 mg/kg via intraperitoneal injection 10 minutes before reperfusion); and (vi) IR+3-TYP group: mice are pretreated with 3-TYP and then subjected to the MI/R operation.

[1]. SIRT3-SOD2-mROS-dependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin. Autophagy. 2015;11(7):1037-51.

[2]. Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3-dependent regulation of oxidative stress and apoptosis. J Pineal Res. 2017 Sep;63(2).

[3]. Identification of a sirtuin 3 inhibitor that displays selectivity over sirtuin 1 and 2. Eur J Med Chem. 2012 Sep;55:58-66.

Cadmium is one of the most toxic metal compounds found in the environment. It is well established that Cd induces hepatotoxicity in humans and multiple animal models. Melatonin, a major secretory product of the pineal gland, has been reported to protect against Cd-induced hepatotoxicity. However, the mechanism behind this protection remains to be elucidated. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd induced mitochondrial-derived superoxide anion-dependent autophagic cell death. Specifically, Cd decreased SIRT3 protein expression and activity and promoted the acetylation of SOD2, superoxide dismutase 2, mitochondrial, thus decreasing its activity, a key enzyme involved in mitochondrial ROS production, although Cd did not disrupt the interaction between SIRT3 and SOD2. These effects were ameliorated by overexpression of SIRT3. However, a catalytic mutant of SIRT3 (SIRT3(H248Y)) lacking deacetylase activity lost the capacity to suppress Cd-induced autophagy. Notably, melatonin treatment enhanced the activity but not the expression of SIRT3, decreased the acetylation of SOD2, inhibited mitochondrial-derived O2(•-) production and suppressed the autophagy induced by 10 μM Cd. Moreover, 3-(1H-1,2,3-triazol-4-yl)pyridine, a confirmed selective SIRT3 inhibitor, blocked the melatonin-mediated suppression of autophagy by inhibiting SIRT3-SOD2 signaling. Importantly, melatonin suppressed Cd-induced autophagic cell death by enhancing SIRT3 activity in vivo. These results suggest that melatonin exerts a hepatoprotective effect on mitochondrial-derived O2(•-)-stimulated autophagic cell death that is dependent on the SIRT3/SOD2 pathway.[1]

---

Sirtuins are a family of highly evolutionarily conserved nicotinamide adenine nucleotide-dependent histone deacetylases. Sirtuin-3 (SIRT3) is a member of the sirtuin family that is localized primarily to the mitochondria and protects against oxidative stress-related diseases, including myocardial ischemia/reperfusion (MI/R) injury. Melatonin has a favorable effect in ameliorating MI/R injury. We hypothesized that melatonin protects against MI/R injury by activating the SIRT3 signaling pathway. In this study, mice were pretreated with or without a selective SIRT3 inhibitor and then subjected to MI/R operation. Melatonin was administered intraperitoneally (20 mg/kg) 10 minutes before reperfusion. Melatonin treatment improved postischemic cardiac contractile function, decreased infarct size, diminished lactate dehydrogenase release, reduced the apoptotic index, and ameliorated oxidative damage. Notably, MI/R induced a significant decrease in myocardial SIRT3 expression and activity, whereas the melatonin treatment upregulated SIRT3 expression and activity, and thus decreased the acetylation of superoxide dismutase 2 (SOD2). In addition, melatonin increased Bcl-2 expression and decreased Bax, Caspase-3, and cleaved Caspase-3 levels in response to MI/R. However, the cardioprotective effects of melatonin were largely abolished by the selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl)pyridine (3-TYP), suggesting that SIRT3 plays an essential role in mediating the cardioprotective effects of melatonin. In vitro studies confirmed that melatonin also protected H9c2 cells against simulated ischemia/reperfusion injury (SIR) by attenuating oxidative stress and apoptosis, while SIRT3-targeted siRNA diminished these effects. Taken together, our results demonstrate for the first time that melatonin treatment ameliorates MI/R injury by reducing oxidative stress and apoptosis via activating the SIRT3 signaling pathway.[2]

C7H6N4

146.1493

146.059

C, 57.53; H, 4.14; N, 38.34

120241-79-4

9833992

White to off-white solid powder

0.866

1

3

1

11

128

0

N1=C(C([H])=NN1[H])C1=C([H])N=C([H])C([H])=C1[H]

VYXFEFOIYPNBFK-UHFFFAOYSA-N

InChI=1S/C7H6N4/c1-2-6(4-8-3-1)7-5-9-11-10-7/h1-5H,(H,9,10,11)

3-(2H-triazol-4-yl)pyridine

3 TYP; 3-TYP; 120241-79-4; 3-TYP; 3-(1H-1,2,3-triazol-4-yl)pyridine; Pyridine(3-TYP); 3-(1H-1,2,3-triazol-4-yl) pyridine; CHEMBL373134; MFCD25956467; Pyridine, 3-(1H-1,2,3-triazol-5-yl)-; 3TYP

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 29~125 mg/mL (198.4~855.3 mM)
Water : ~1.3 mg/mL (~8.6 mM)
Ethanol : 16.7~29 mg/mL (114.1~198.4 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (14.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (14.23 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (14.23 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)