Main Research Findings
1) Intra-articular supplementation with GHK-CU was found to improve graft healing following anterior cruciate ligament reconstruction in rats.
2) GHK-Cu was found to elicit a protective effect in lipopolysaccharide-induced acute lung injury through the inhibition of excessive inflammatory responses.
Selected Data
1) This study performed by the research team of Fu et al investigated the effects of the tripeptide-copper complex GHK-Cu on healing outcomes in a rat model of anterior cruciate ligament reconstruction (ACLR). Seventy-two male Sprague-Dawley rats, approximately 12 weeks old and weighing 418.2 ± 22.2g, were used in the study. Unilateral ACLR was performed on each rat’s right knee, a procedure detailed in previous research by the authors. The surgical technique involved using the ipsilateral flexor digitorum longus tendon to reconstruct the ACL. This tendon graft was carefully inserted into femoral and tibial bone tunnels, each 1.1 mm in diameter, and secured to the periosteum with 4N graft tensioning, achieved using a freely suspended weight [1].
Following ACLR, rats were randomly assigned to one of three groups including: a saline group that received intra-articular injections of saline; a low-dose GHK-Cu group that received intra-articular injections of 0.3 mg/ml GHK-Cu; and a high-dose GHK-Cu group, that received intra-articular injections of 3 mg/ml GHK-Cu.
GHK-Cu was prepared by dissolving GHK acetate and copper (II) chloride 2-hydrate in 0.9% saline. The resulting 10mg/ml GHK-Cu stock solution was filtered and aliquoted for storage until use. To prevent swelling, intra-articular injections were administered weekly from the second to the fifth week post-operatively. Each injection involved loading 50 µl of the treatment solution into a syringe with a 26G removable needle. Under general anesthesia, the needle was inserted from the medial side of the knee, with the knee extended, positioning it at the bottom of the patella to access the joint space. The solution was then injected slowly.
Rats were euthanized at two primary time points: 6 weeks and 12 weeks post-operation. At these times, knee specimens were harvested for various analyses. A range of biomechanical, histological, and functional assessments were performed, first an anterior-posterior (A-P) knee laxity test that was conducted at 6 and 12 weeks post-operation using a previously established protocol. Freshly trimmed tibia and femur shafts were embedded in adhesive polymer in plastic tubes, ensuring axial alignment to preserve the natural varus/valgus angle. The specimens were then mounted on a mechanical testing machine with a 50N load cell. The knee was positioned at 70° flexion. A posterior displacement of 0.5 mm was applied, followed by resetting to force zero, and then an anterior displacement of 1 mm, returning to the starting position. This cycle was repeated four times at 40 mm/min. Static A-P knee laxity was measured as the total displacement caused by a fixed anterior and posterior loading (0.5N) at the neutral position [1].
Following the laxity test, specimens were stored at -20°C and thawed for 2 hours at room temperature. The femur-graft-tibia complexes were vertically aligned, and tensile tests were performed at a crosshead speed of 40 mm/min with a 50N load cell until graft failure was detected by an abrupt drop in loading. Failure load, mode of failure, and the stiffness (slope of the force-displacement curve) of the complex were recorded. Next, at 6 and 12 weeks post-operation, euthanized rats had their knee joints fixed, decalcified, and paraffin-embedded. Five-micrometer-thick paraffin sections were collected along the sagittal plane of the knee. Sections from femoral tunnels, intra-articular graft mid-substance, and tibial tunnels were selected for H&E staining, and epiphyseal regions were also examined. Sections were examined under bright fields and polarized illumination. Histological evaluation was performed by two independent, blinded examiners using a developed scoring system based on matrix degeneration of the tendon graft and healing responses at the graft-tunnel interface [1].
Finally, gait analysis was performed pre-injury, and at 6 and 12 weeks post-operation using Catwalk XT 9.0 technology. This assessed functional recovery and potential pain-associated gait changes. Recorded runs with a steady walking speed, with a variation <30%, were accepted, and three to five runs were used for the calculation of gait parameters. A Limb Idleness Index (LII) > 1.3 indicated pain-associated gait changes [1].
2) This study performed by the research team of Park et al investigated the protective effects of the tripeptide-copper complex GHK-Cu against lipopolysaccharide (LPS)-induced acute lung injury (ALI), employing both in vitro macrophage cell cultures and an in vivo mouse model. The core materials for the study included GHK-Cu, and key fluorescent probes and antibodies were also critical components. 2′,7′-dichlorofluorescin-diacetate (DCFDA) was used for reactive oxygen species (ROS) measurement. A comprehensive panel of antibodies was utilized for Western blotting, targeting phosphorylated and total forms of ERK1/2, p38 MAPK, JNK1/2, and NF-κB p65 (Ser536), with β-actin serving as a loading control. An additional NF-κB p65 antibody for immunostaining was purchased from Santa Cruz Biotechnology [2].
For the in vitro experiments, a murine macrophage cell line RAW 264.7 was used. Peritoneal macrophages were prepared by injecting 3% thioglycollate intraperitoneally into mice, harvesting the cells four days later. Both RAW 264.7 cells and peritoneal macrophages were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 5% CO₂, and 95% air at 37°C. For cell stimulation, cells were pretreated with various concentrations of GHK-Cu of 1, 5, or 10 µM for 18 hours, followed by stimulation with 100 ng/ml LPS for specific time periods. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. Intracellular ROS levels were detected by incubating cells with 10 µM CM-DCFDA for 30 minutes, then analyzed by Accuri-C6 flow cytometry. Superoxide dismutase (SOD) activity was quantified using a commercial kit, based on the inhibition of WST-1 formazan dye generation. Nitric oxide (NO) secretion was measured using the Griess reagent [2].
Enzyme-linked immunosorbent assay kits were used to determine the levels of pro-inflammatory cytokines, specifically TNF-α and IL-6, in cell culture supernatants. Immunostaining was performed by seeding cells on glass coverslips, fixing with paraformaldehyde, permeabilizing with Triton X-100, and then incubating with primary anti-NF-κB p65 antibody, followed by Alexa Fluor 546-conjugated secondary antibody. Images were captured using a confocal laser-scanning microscope to assess NF-κB p65 nuclear translocation. Western blot analysis involved preparing whole cell lysates using RIPA lysis buffer, quantifying protein concentration via BCA assay, followed by SDS-PAGE, transfer to nitrocellulose membranes, incubation with primary and HRP-conjugated secondary antibodies, and detection using an ECL solution.
The in vivo model utilized 10-week-old male C57BL/6 mice that were housed in environmentally controlled conditions with a 12-hour dark-light cycle, receiving standard laboratory chow and water ad libitum. ALI was induced via intratracheal administration of 1 µg/g LPS dissolved in 50 µl of saline. For GHK-Cu pretreatment, mice received intraperitoneal injections of GHK-Cu of 1 or 10 µg/g every 24 hours for three days prior to LPS administration. Control groups received saline injections. Mice were sacrificed 24 hours post-LPS administration. Histopathological analysis involved fixing left lung lobes in 10% neutral formalin, embedding in paraffin, sectioning into 4 µm thick sections, and staining with hematoxylin and eosin (H&E). Lung injury was graded from 0 (normal) to 4 (severe) based on neutrophil infiltration, congestion, edema, and alveolar wall thickness. Lungs were harvested, and bronchoalveolar lavage fluid (BALF) was collected by lavaging the trachea twice with 1 ml of ice-cold PBS. BALF was centrifuged to collect supernatants for cytokine and total protein content analysis, while cell pellets were used for differential cell counts via cytospin centrifuge and Hema-3 staining. Myeloperoxidase (MPO) activity, a marker for neutrophil infiltration, was measured in lung tissue homogenates using a previously described spectrophotometric method, assessing H₂O₂-dependent o-dianisidine oxidation. Glutathione (GSH) levels in lung homogenates were determined enzymatically, and NO detection was performed using Griess reagent [2].
Discussion
1) The results of the study performed by the research team of Fu et al demonstrated a transient improvement in graft healing outcomes following ACLR in rats treated with GHK-Cu, primarily observed at 6 weeks post-operation. Initial observations regarding the animal model indicated that out of 72 operated rats, 3 died during anesthesia and were subsequently replaced. Further surgical complications such as fixation failure, sub-optimal tunnel placement, and post-surgical issues like wound re-sutures, patella dislocation, and infection led to the exclusion of some rats from statistical analyses for gait and mechanical test data. This ensured that only animals with successful surgical outcomes and no confounding complications were included in the primary data analysis [1].
A key assessment involved the A-P knee laxity test. At 6 weeks post-operation, rats in both the 0.3 mg/ml and 3 mg/ml GHK-Cu treatment groups exhibited a significantly smaller side-to-side difference in A-P knee laxity compared to the saline group. This suggests that GHK-Cu treatment, particularly the lower dose, contributed to better knee stability in the early healing phase. However, this beneficial effect was not sustained, as no significant difference in A-P laxity was found between any of the groups at the 12-week post-operation time point. This transient nature of the GHK-Cu effect indicated that its beneficial impact diminished as treatment was discontinued and healing progressed.
The load-to-failure test provided further mechanical insights into graft healing. While there was no significant difference in the ultimate load of the graft complex between GHK-Cu and saline groups at either 6 or 12 weeks post-operation, a notable finding emerged regarding graft stiffness. The graft complex in the 0.3 mg/ml GHK-Cu group demonstrated significantly higher stiffness compared to the saline group at 6 weeks post-operation. This increased stiffness suggests an improved mechanical integrity of the graft during the early healing period with lower-dose GHK-Cu. Similar to laxity, this enhanced stiffness was not maintained at 12 weeks post-operation. Consistently across all groups and time points, every graft failed mid-substance during the pull-out test, indicating that the graft itself, rather than the fixation points, remained the weakest link [1].
Histological analysis offered a microscopic view of the healing process. At 6 weeks post-operation, better graft incorporation at the tibial tunnel was observed in the 0.3 mg/ml GHK-Cu group, though this difference did not reach statistical significance. A complete bony layer surrounding the graft at the tibial tunnel was evident in GHK-Cu treated groups administered both 0.3 mg/ml and 3 mg/ml, contrasting with a “leaky” tunnel interface often seen in the saline group, which pointed towards better bone healing. Despite this qualitative observation, the overall bone healing score was not statistically different [1]. Furthermore, at 12 weeks post-operation, no significant histological differences were found between any of the groups. In both 6- and 12-week time points, graft degeneration, characterized by reduced collagen birefringence, was less severe in the tibial tunnel and intra-articular mid-substance compared to the femoral tunnel. The 0.3 mg/ml GHK-Cu group also exhibited reduced graft degeneration compared to controls at 6 weeks, though not statistically significant. Image analysis revealed increased cellularity in GHK-Cu treated samples at the intra-articular mid-substance at 6 weeks, particularly with the higher dose, though this difference was not statistically significant [1].
Finally, gait analysis, performed using the Catwalk XT system, assessed the functional recovery of the rats. Weekly intra-articular injections of GHK-Cu, compared to pre-injury levels, caused the injured limb to become significantly “idled” in rats undergoing ACLR and ipsilateral flexor tendon donor site injury. Parameters such as target print ratio (TPR), swing duration ratio (SWR), and the Limb Idleness Index (LII) were significantly altered over time, reflecting decreased loading and increased paw elevation time on the operated hindlimb. Despite these changes, no significant differences were observed among the experimental groups for any of the gait parameters. This indicated that while the injured limb showed adaptive gait changes, GHK-Cu treatment did not differentially improve the overall gait mechanics compared to saline. At 6 weeks post-operation, 30% of rats in every group developed an LII > 1.3, signifying pain-associated gait changes. This percentage remained similar for saline and 0.3 mg/ml GHK-Cu groups at 12 weeks, but increased to 50% in the 3 mg/ml GHK-Cu group, though this difference was not statistically significant. In conclusion, the study found that GHK-Cu offered transient mechanical and histological benefits at 6 weeks post-ACLR, but these effects did not persist at 12 weeks, and no significant functional gait improvements were observed between treated and untreated groups [1].
Figure 1: Changes in A) anchor print ratio, B) target print ratio, C) swing duration ratio, and D) limb idleness index across the three treatment groups at pre-injury, 6 weeks post-operative, and 12 weeks post-operative.
2) The results of the study performed by the research team of Park et al comprehensively detail the protective effects of GHK-Cu against LPS-induced inflammation and ALI, beginning with in vitro*experiments on RAW 264.7 macrophages and extending to an in vivo mouse model. In RAW 264.7 macrophages, GHK-Cu significantly mitigated the LPS-induced increase in ROS production. While LPS exposure alone resulted in a 59% increase in oxidized DCF (a measure of ROS) compared to control cells, pretreatment with GHK-Cu at concentrations of 1, 5, and 10 µM significantly decreased ROS production. Concurrently, LPS treatment led to a significant decrease of approximately 19% in SOD activity, a crucial antioxidant enzyme. GHK-Cu pretreatment, across the same concentration range, successfully restored SOD activity to near control levels, highlighting its antioxidant-boosting capabilities. Importantly, GHK-Cu did not negatively impact cell proliferation or NO secretion in these macrophages, suggesting its protective effects were specific to inflammatory pathways rather than general cytotoxicity [2].
Further in vitro analysis revealed GHK-Cu’s capacity to attenuate the release of key pro-inflammatory cytokines. Exposure of RAW 264.7 cells to LPS for four hours dramatically increased the secretion of IL-6 to 613.2 ± 35.1 pg/ml and TNF-α to 1556.3 ± 23.3 pg/ml. However, pretreatment with 10 µM GHK-Cu significantly reduced the secretion of both TNF-α and IL-6 to much lower levels, confirming its anti-inflammatory properties.
Investigating the underlying molecular mechanisms, GHK-Cu was found to block critical signaling pathways. LPS stimulation significantly induced the phosphorylation of NF-κB p65 at Ser536 and promoted its nuclear translocation. GHK-Cu treatment with 1, 5, and 10 µM effectively inhibited both the LPS-stimulated phosphorylation of NF-κB p65 and its subsequent nuclear translocation, thereby suppressing NF-κB activation. This is crucial as NF-κB plays a pivotal role in regulating pro-inflammatory gene expression. Moreover, GHK-Cu significantly inhibited the LPS-induced phosphorylation of p38 MAPK and slightly decreased the phosphorylation of JNK1/2, while having no observable effect on ERK1/2. These findings suggest that GHK-Cu’s anti-inflammatory action is mediated, at least in part, by modulating the p38 MAPK and NF-κB signaling pathways.
Moving to the in vivo mouse model of LPS-induced ALI, GHK-Cu demonstrated significant protective effects against lung damage. LPS administration caused characteristic morphological changes in lung sections, including severe immune cell infiltration, interstitial edema, alveolar wall thickening, and hemorrhage, indicative of successful ALI induction. While GHK-Cu treatment alone did not cause any visible histological changes or toxicity, pretreatment with GHK-Cu markedly attenuated these pathological alterations, resulting in a significantly reduced lung injury score compared to the LPS-only group. This macroscopic observation underscored GHK-Cu’s ability to protect lung tissue from acute inflammatory insult [2].
Consistent with the in vitro findings, GHK-Cu increased antioxidant enzymes and decreased pro-inflammatory cytokines in vivo. LPS administration in mice resulted in decreased SOD activity and total glutathione GSH in lung homogenates. However, pretreatment with 10 µg/g GHK-Cu significantly increased both SOD activity and GSH levels, bringing them to values comparable to control mice. Concurrently, LPS significantly elevated TNF-α and IL-6 levels in BALF, which were markedly decreased in mice pretreated with 10 µg/g GHK-Cu. These results confirm GHK-Cu’s role in bolstering antioxidant defenses and suppressing systemic inflammatory responses in the lung [2].
Figure 2: Changes in A) SOD activity, B) total GSH, C) IL-6 levels in BALF, and D) TNF-alpha levels in BALF across the treatment groups in response to varying doses of GHK-Cu.
Furthermore, GHK-Cu effectively reduced immune cell infiltration and alveolar permeability, critical hallmarks of ALI. MPO activity, a marker for neutrophil presence, and neutrophil counts in the lung were significantly increased by LPS. GHK-Cu pretreatment showed a dose-dependent trend towards lower MPO activity and neutrophil infiltration. The total cell counts and total protein concentration in BALF, indicators of cellular infiltration and increased alveolar permeability, respectively, were significantly elevated by LPS. Again, 10 µg/g GHK-Cu pretreatment significantly decreased both total cell counts and total protein in BALF, demonstrating its ability to maintain alveolar-capillary membrane integrity and reduce inflammatory cell accumulation.
Finally, the in vivo mechanistic studies confirmed the modulation of MAPK and NF-κB signaling pathways. LPS administration induced significant phosphorylation of NF-κB p65 (Ser536), p38 MAPK, and JNK1/2 in lung homogenates. Pretreatment with 10 µg/g GHK-Cu significantly reduced the phosphorylation of both NF-κB p65 and p38 MAPK, and slightly decreased JNK1/2 phosphorylation, with no effect on ERK1/2. These in vivo results align with the in vitro data, consolidating the understanding that GHK-Cu exerts its protective effects in ALI by inhibiting excessive inflammatory responses through its anti-inflammatory and antioxidant properties, mediated by the suppression of NF-κB and p38 MAPK signaling pathways [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).
Citation
[1] Fu SC, Cheuk YC, Chiu WY, Yung SH, Rolf CG, Chan KM. Tripeptide-copper complex GHK-Cu (II) transiently improved healing outcome in a rat model of ACL reconstruction. J Orthop Res. 2015;33(7):1024-1033. doi:10.1002/jor.22831
[2] Park JR, Lee H, Kim SI, Yang SR. The tri-peptide GHK-Cu complex ameliorates lipopolysaccharide-induced acute lung injury in mice. Oncotarget. 2016;7(36):58405-58417. doi:10.18632/oncotarget.11168
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