Curcumin

Curcumin was purchased from Sigma-Aldrich Company (CAS Number: 458-37-7), Stock of Curcumin solution (5 mg/mL) was prepared by dissolving Curcumin powder in Dichloromethane (20 mL). One ml of stock solution was added to boiling water (50 mL) in a drop-wise manner under ultra-sonication conditions (XUBA3Analogue Ultrasonic Bath, Grant Company) with an ultrasonic power and frequency of 50 kHz. The solution was sonicated for about 30 min. After sonication, the mixture was stirred at 800 rpm for 20 min till the orange-coloured precipitate was obtained. Thereafter, the supernatant was discarded, and the pellet obtained was used for further study [15 ].

All animal studies were approved by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee. Destabilization of the medial meniscus (DMM) was established in adult C57BL/6 male mice (male, 5–6 months of age) by surgically transecting the medial meniscotibial ligament (MMTL) in the right hind limb [32 (link)]. Briefly, the joint capsule immediately medial to the patellar tendon was incised, followed by blunt dissection of the infrapatellar fat pad, to provide visualization of the MMTL of the medial meniscus. The MMTL was transected, leading to destabilization of the medial meniscus (DMM). In the sham surgery, the MMTL was visualized but not transected. The joint capsule and skin were closed by suture. Immediately after the DMM surgery, mice were subjected to (1) oral administration of 50 mg/kg curcumin (Sigma) dissolved in corn oil or vehicle (corn oil only) administered via oral gavage (n = 8/group), or (2) topical application of curcumin nanoparticles (0.07 mg of 10 μg curcumin/1 mg nanoparticles) or vehicle control (coconut oil) on the skin, within a 5-mm² area directly above the DMM-operated knee (n = 5/group), once daily for 8 weeks.

In order to establish the starved condition and induce autophagy, cells were incubated in Earle’s balanced salt solution (EBSS; WelGENE Inc.) for 4 h. For treatment of curcumin, cells were plated at a density of 1 × 10⁵ cells·mL⁻¹. 24 h after cell plating, cells were treated with curcumin (Sigma‐Aldrich, Saint Louis, MO, USA) diluted in dimethyl sulfoxide (DMSO; Sigma‐Aldrich) for 6 h. MDA‐MB‐231/A cells were treated with 50 μm curcumin, and MDA‐MB‐231/A tumorspheres were treated with 25 μm curcumin. MDA‐MB‐231 cells were treated with 30 μm curcumin. Rapamycin (Sigma‐Aldrich) was used to induce autophagy and treated at 100–500 nm concentration for 24 h. To inhibit the autophagosome–lysosome fusion [17 (link)], chloroquine (CQ; Sigma‐Aldrich) was diluted in DMSO, and the cells were treated with 10 μm chloroquine for 3 h.

The rats were randomly equally divided into three groups (n = 10 for each group):

I/R-Curcumin group (Cur) in which the rats were subjected to renal ischemia for 45 min and precondition with 60 mg/kg (i.p. injection) of Curcumin (Sigma, St. Louis, MO) at 45 min before I/R induction; The dosage of Curcumin was in line with previous study [7 (link)].

I/R-vehicle group (IRI), in which the rats were subjected to renal ischemia for 45 min and precondition with 60 mg/kg (i.p. injection) of saline (Sigma, St. Louis, MO) at 45 min before I/R induction.

Sham-operated group (Sham), in which the rats were subjected to identical surgical procedure as above but without renal I/R injury.