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Di-melittin (anti-inflammatory peptide)

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• Name: Di-melittin
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Background

Melittin, as a crucial active component of bee venom, has garnered significant attention in the scientific community for its remarkable anti-inflammatory and immunomodulatory properties. The molecular structure of melittin enables it to effectively intervene in and regulate the human immune response, demonstrating substantial potential in studies of various inflammatory diseases.

In recent years, researchers have gradually unveiled the possible mechanisms of melittin in the treatment of inflammatory bowel disease (IBD) through multiple laboratory studies and animal model experiments. melittin can reduce inflammatory responses by inhibiting the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Additionally, it modulates the activity of immune cells, particularly regulatory T cells and macrophages, thereby maintaining balance within the immune system. These mechanisms work synergistically to significantly alleviate intestinal inflammation and aid in restoring normal intestinal function.

In terms of clinical application, melittin shows promise in improving IBD symptoms. However, further research is needed to optimize its safety and efficacy.

The anti-inflammatory principle of melittin

Anti-inflammatory effect

Figure 1:Anti-inflammatory effect of melittin

Reference: Liu Minchen & Du Ruofei, Chinese Journal of Modern Applied Pharmacy, 2023

In terms of anti-inflammatory effects, melittin plays a crucial role in regulating inflammatory signaling pathways. It inhibits the activity of the NF-κB pathway, reducing the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Melittin can suppress the phosphorylation of IKK and IκB, thereby blocking the nuclear translocation of NF-κB and interfering with inflammatory signal transduction. Additionally, melittin affects the MAPK signaling pathway by inhibiting the phosphorylation of JNK and p38, thus regulating the production of pro-inflammatory cytokines and reducing the activity of inflammatory cells and the secretion of inflammatory mediators. Furthermore, melittin interferes with the JAK/STAT pathway, inhibiting the activity of STAT transcription factors decreasing the secretion of inflammatory cytokines, which helps alleviate inflammatory diseases. Melittin also reduces inflammation by inhibiting Akt phosphorylation, which interferes with the expression of inflammatory proteins and suppresses the production of inflammatory mediators such as COX-2, iNOS, and cPLA2. Additionally, melittin promotes the recruitment of immune cells to the site of inflammation and induces T-cell apoptosis, thereby modulating the immune response and further alleviating inflammation.

Antibacterial effect of melittin

Figure 2:Effect of melittin on cell membrane

Reference: Zhang HQ, Sun C, Xu N, Liu W. The current landscape of the antimicrobial peptide melittin and its therapeutic potential. Front Immunol. 2024 Jan 22;15:

Melittin monomers can attach to the membrane surface and spontaneously integrate into natural or artificial phospholipid bilayers, thereby reducing the rigidity between the polar and non-polar regions and decreasing the permeability barrier. It has been proposed that melittin-induced membrane permeability may result from the formation of toroidal pores or fissures within the membrane.

Figure 3:Anti-fungal mechanisms of melittin

Reference: Memariani, H., & Memariani, M. (2020). Anti-fungal properties and mechanisms of melittin. Applied microbiology and biotechnology, 104(15), 6513–6526.

Melittin affects fungal cells through multiple mechanisms. It externalizes phosphatidylserine and forms cyclic pores, disrupting cell membrane integrity and increasing membrane permeability, leading to an imbalance of intracellular and extracellular substances, such as potassium ion leakage resulting in ionic imbalance. Additionally, Melittin induces apoptosis via a reactive oxygen species (ROS)-mediated mitochondrial/caspase-dependent pathway, inhibiting fungal growth. It also inhibits the activity of (1,3)-β-D-glucan synthase, affecting cell wall stability. Furthermore, Melittin can alter fungal gene expression and cause DNA fragmentation, further disrupting growth and reproduction.

Functional Evolution Design of Melittins

Modification Method

The toxicity of melittin limits its clinical applications, so we have ingeniously modified and optimized the melittin molecule. Specifically, we employed an innovative approach by linking two melittin monomers with a carefully designed linker. This design allows the two peptide monomers to form a stable hairpin structure.

https://static.igem.wiki/teams/5332/registry/melittin/rmelittin4.png4

Figure 4：Amino acid sequence and nucleic acid sequence

Figure 5：Possible structural diagram

Modification Results

This modification effectively reduces the cytotoxicity of melittin while significantly enhancing its function as an immune stimulant. This improvement not only increases the safety of melittin in biomedical applications but also enhances its potential value in immunotherapy. The dual optimization allows melittin to be applied in a broader range of clinical settings, offering more ideal therapeutic options and higher treatment efficiency.

Test

Peptide cell experiment

Another drug named Indo was also tested, but it is only shown for comparison here. Constructing the cell model: Thaw macrophages and passage them twice before seeding them into a 24-well plate. Once the cells reach confluence again, subject them to the same starvation treatment. After infection procedures, treat the experimental groups with different concentrations of Mel and Indo. Collect the cells after four hours of incubation.

Figure6：Cellular Reactive Oxygen Species (ROS) Levels

Using flow cytometry to measure reactive oxygen species (ROS) levels, as shown in Figure 6, the experimental groups treated with 5, 10, and 15 ppm Mel showed a significant decrease in ROS levels. In contrast, applying different concentrations of the peptide to uninfected macrophages indicated that Mel does not reduce ROS levels in healthy cells (Figure 6). These results suggest that appropriate concentrations of Mel can significantly reduce ROS content in inflammatory cells.

Figure 7：Expression Levels and Changes of Four Inflammatory Factors

Using RT-qPCR, the expression levels and changes of four inflammatory factors were measured, as shown in Figure 7. Mel at a concentration of 15 ppm significantly upregulated all four inflammatory factors, while a concentration of 10 ppm significantly downregulated the expression levels of IL-1β and IL-6. We speculate that appropriate concentrations of both peptides can downregulate certain inflammatory factors. However, excessively high concentrations may cause the peptides to act as antigens, stimulating macrophages to produce an inflammatory response, thereby significantly increasing the expression levels of inflammatory factors.

Figure 8:Post-Infection Cell Count and Intracellular Viable Bacteria Count As shown in Figure 8, only the cells treated with 5 and 10 ppm Mel exhibited a significant reduction in intracellular bacteria, while the other groups showed no noticeable change.

In vivo peptide experiments

Firstly, a mouse intestinal inflammation model was constructed. A batch of female Balb/cJNju-Foxn1nu/Nju Hfh11nu mice were ordered and experimental procedures were carried out according to the table and all fluids were administered into the mice by gavage. The concentration of Salmonella is , Mel and Indo is 10ppm. The intestinal specimens taken were from the cecum to the colon.

Figure 9:Each group's colon length in mice

The colon length in mice was used to characterize the level of intestinal inflammation. Results showed that both Mel and Indo significantly alleviated the shortening of colon length due to inflammation, with Mel demonstrating a superior effect.

Figure10:Colon tissue histological examination

After dehydration, embedding, and sectioning, colon tissues were stained with H&E. As shown in Figure 10, the S.Tm group exhibited damaged intestinal epithelial cells and compromised mucosal integrity compared to the PBS group. The symptoms were notably alleviated in the treatment groups, with the Mel group showing superior results.

Figure 11:Immunofluorescence staining

Some tissues were fixed with 4% paraformaldehyde and subjected to immunofluorescence staining. As shown in Figure 11, the IL-6 fluorescence signal intensity in tissues treated with Salmonella was significantly higher than in the PBS group, indicating severe intestinal inflammation. In contrast, the Mel group showed significantly lower IL-6 fluorescence intensity compared to the S.Tm group, suggesting that Mel has an anti-inflammatory effect.

Figure 12:Immunofactor and Immune Cell Marker Protein Staining

Colon tissues were sectioned and fully dissociated for staining of immunofactors and immune cell marker proteins, followed by flow cytometry to measure expression levels. As shown in Figure 12, Mel significantly reduced the levels of IFN-γ, IL-6, TNF-α, F4/80, and Ly6G in the colon tissues of mice with colitis.

Figure 13：Expression levels of inflammatory factors

Extract a portion of colon tissue and isolate cellular RNA for RT-qPCR to assess the expression levels of various inflammatory factors. As shown in Figure 3E, the expression levels of IL-6, IL-1β, and IL-8 in the intestinal tissue treated with Mel were significantly reduced compared to the control group.

Mathematical Modeling Results

We used an agent-based model to simulate the effects of melittin (Mel) and indolicidin (Indo) on immune cells, cytokine levels, and colon length in mice. The simulation ran for 7 days, with iterations every half day. The main goal was to observe the recovery and damage of colon tissue by simulating the immune system's response to different drugs.

<img src="rmelittin14.png" style="width: 500px; height: 300px;">

Fiugre 14:Changes in multiple physiological indicators over time after adding melittin and indolicidin during colitis.

Changes in colon length are shown in Figure 14(A). Before t=6, colon length significantly shortened due to colitis, indicating a typical inflammatory response. After treatment, the colon length recovered more quickly with melittin (Mel), suggesting it may have a better effect on colon repair.

The number of immune cells is shown in Figure 14(B). Before t=6, the number of three types of immune cells increased to varying degrees due to colitis. After treatment, the number of immune cells significantly decreased with melittin (Mel).

Cytokine levels are shown in Figure 14(C). Before t=6, both anti-inflammatory and pro-inflammatory cytokines increased due to inflammation. After treatment, cytokine levels began to decrease with the addition of melittin (Mel).

References