Exploring the Beneficial Effects of GHK-Cu on an Experimental Model of Colitis and the Underlying Mechanisms

Jing Yao^1*Shuzhen Mao²Jiahui Huang¹Junyan Li³Fang Sun¹Qilian Zhang⁴Qing Cheng¹Wei Zeng¹Dongya Lei¹Shiyan Wang¹

• ¹Jining Key Laboratory of Pharmacology, School of Basic Medicine, Jining Medical University., Jining, Shandong, China
• ²Department of Pharmacy, Shandong First Medical University & Shandong Academy of Medical Sciences., Jinan, Shandong., China
• ³Department of Pathology, Second People's Hospital of Ningyang, Taian, Shandong, China
• ⁴Department of Pathology, People's Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China

Ulcerative colitis (UC) is a chronic inflammatory disorder of the gastrointestinal tract, leading to significant clinical symptoms and a reduced quality of life. Glycyl-L-histidyl-L-lysine-copper (GHK-Cu) is known for its potent anti-inflammatory and antioxidant properties. Nonetheless, the therapeutic potential and precise mechanisms of GHK-Cu in UC remain largely unknown. This study found that GHK-Cu demonstrated significant therapeutic effects in dextran sulfate sodium (DSS)-induced UC in mice. It alleviated weight loss, improved the disease activity index (DAI), reduced colonic edema and shortening. The results of hematoxylin-eosin and Alcian blue-periodic acid Schiff staining showed that GHK-Cu attenuated inflammatory damage and increased goblet cell numbers. Western blots results showed that GHK-Cu suppressed inflammatory cytokines such as TNF-α, IL-6, and IL-1β, and promoted mucosal repair. Additionally, a co-culture system of mouse colonic epithelial cells (MCECs) and mouse peritoneal macrophages (MPMs) revealed that GHK-Cu facilitated MCECs healing, impaired by DSS, by upregulating zonula occludens-1 (ZO-1) and Occludin expression. Subsequently, network pharmacology and molecular docking identified NAD-dependent deacetylase sirtuin-1 (SIRT1) as a potential target of GHK-Cu. Western blots results showed that GHK-Cu upregulated SIRT1 protein expression and suppressed the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in colon tissue and MCECs of the co-culture model. Moreover, STAT3-targeting siRNA (siSTAT3) was utilized to explore the mechanism of GHK-Cu in the co-culture model and lipopolysaccharide (LPS)-induced MPMs. Our findings revealed that after transfection with siSTAT3, the stimulant effect of GHK-Cu on the healing of MCECs and the effect on the protein expression of ZO-1 and Occludin is canceled. Nevertheless, after transfection with siSTAT3, it could inhibit the expression of inflammatory factors in conjunction with GHK-Cu. This indicates that GHK-Cu may promote mucosal healing and enhance tight junction protein expression by regulating the SIRT1/STAT3 pathway. In addition to suppressing p-STAT3 expression, GHK-Cu may utilize additional pathways to inhibit inflammatory factors. Furthermore, we found that GHK-Cu could inhibit retinoic acid receptor-related orphan receptor gamma t (RORγt) expression in the colon tissue of UC mice, suggesting that GHK-Cu may reduce the number of Th17 cells. In summary, GHK-Cu may treat UC by acting on the SIRT1/STAT3 pathway.