Casp 3 c

With the aid of a thiobarbituric acid assay, the content of malondialdehyde (MDA), an end product of lipid peroxides, was assessed in the testes, as described in [61 (link)], and the final absorbance was recorded at 535 nm. Rat-specific ELISA kits for SIRT1 (AFG Bioscience, Northbrook, IL, USA, Cat. no. EK720561), Nrf2 (Cat. no. MBS012148), and HO-1 (Elabscience, Wuhan, China, Cat. no. E-EL-R0488) [62 (link)] were used for the assay of each corresponding target. The Nrf2 content was examined in the testicular nuclear extract that was prepared with the aid of a Cayman nuclear extraction kit (Cayman Chemical, Ann Arbor, MA, USA, Cat. No. 10009277). The final absorbance measurement for SIRT1, Nrf2, and HO-1 was applied at 450 nm. The activity of GPx was attained using a Sigma-Aldrich colorimetric kit (Sigma-Aldrich, St. Louis, MO, USA, Cat. no. CGP1). To this end, monitoring the absorbance decline was applied at 340 nm using a kinetic program. Testicular caspase-3 activity was measured with the aid of the corresponding Sigma-Aldrich colorimetric assay kit according to the manufacturer’s instructions (Cat. no. CASP-3-C; Sigma-Aldrich, USA). At 405 nanometers, the O.D. of the final color was read.

The ATO‐induced cardiomyocytes apoptosis was assessed by using Hoechst 33342/annexin V staining. The cardiomyocytes were cultured in 6‐well plates for 24 h. After designated treatment, the cells were washed with annexin V binding buffer, incubated with annexin V‐FITC for 15 min at room temperature, and then counterstained with Hoechst 33342 for nuclei, after washing twice with PBS, the images of apoptotic cells were taken on a fluorescence microscopy. Caspase‐3 activation was measured by caspase 3 colorimetric assay kit (CASP‐3‐C, sigma) which is based on the hydrolysis of the peptide substrate acetyl‐Asp‐Glu‐Val‐Asp p‐nitroanilide (Ac‐DEVD‐pNa) by caspases‐3, resulting in the release p‐nitroaniline (pNA) moiety. Absorbance of pNA in each designated group as mentioned above is measured at 405 nm wavelength in ELISA reader using 96 well plate microassay method, according to the manufacture's instruction.

The activity of caspase-3 enzymes was measured in cell lysates by a colorimetric assay kit (CASP-3-C, SIGMA-ALDRICH, Saint Louis, MO, USA), that is based on the hydrolysis of the compound acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) (A 2559, Sigma-Aldrich, Saint Louis, MO, USA) by caspase-3. The reaction releases the p-nitroaniline moiety (p-NA) that absorbs at 405 nm. Cells were pre-incubated with 5 μΜ doxorubicin hydrochloride (DOX) (Sigma–Aldrich, Deisenhofen, Germany) for 3 h for caspase cascade initiation [41 (link),42 (link)]. Samples (10 μL) were tested both with and without the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-al (Ac-DEVD-CHO, 20 μΜ final concentration), in a total reaction volume of 100 μL in 96-well plates. The substrate Ac-DEVD-pNA concentration was 200 μM and the assay was performed at 37 °C for 90 min. The caspase-3 specific activity was expressed as the percentage of μmol of p-nitroaniline released per min per μg of total protein values compared to control.

The inhibitory activity of the top four plants resulted from network pharmacology analysis on caspase 3 gene was quantified as illustrated on caspase 3 colorimetric assay kit (CASP-3-C)¹⁰⁸ supplied by Sigma, and described by Ros et al.^{109 (link)} as in supplementary materials.

Caspase activity was measured in liver homogenate using a commercial kit (CASP3C, Sigma-Aldrich, Madrid, Spain) according to the manufacturer’s instructions.