*4.2. Prevents Amyloid-*β*-Induced Toxicity*

Amyloid-β is a key component of drusen deposits that are formed in the AMD retinas. Administration of Humanin G reduces amyloid-βloads and inhibits amyloid-β-induced cell apoptosis by (a) restoring amyloid-β-mediated decline in calcium homeostasis, (b) suppressing amyloid-β-induced membrane fluidity changes, (c) restoring mitochondrial membrane potential, and (d) decreasing intracellular reactive oxygen species [43]. This finding is crucial as Humanin G's ability to mitigate amyloid-β-induced cytotoxicity might be used as a therapeutic approach to delay the progression of dry AMD.

### *4.3. Stress Resistance Against ER Stress-Induced Apoptosis*

Humanin exerts its therapeutic benefits by antagonizing the action of a plethora of cellular insults, thereby protecting the RPE cells against cytotoxicity. Treatment with Humanin downregulates the expression of an ER stress marker CHOP (C/EBP Homologous Protein), inhibits ROS (Reactive Oxygen Species) production, and regulates intracellular calcium influx, thereby preventing RPE cell apoptosis [44,45]. Treatment of primary human RPE cells with three ER stress sensors, i.e., Tunicamycin, Brefeldin A, and Thapsigargin, induced mitochondrial damage and dose-dependent loss of RPE cells. However, pretreatment with Humanin provided significant cytoprotection against ER stress-induced cell death by restoring the depleted mitochondrial glutathione (GSH) levels to normal, reducing mitochondrial superoxide generation, and downregulating ER stress-induced apoptotic Caspase 4 and Caspase 3 [46].

### *4.4. Activation of the ERK, AKT, and STAT3 Signaling Pathways*

As a secretory peptide, Humanin regulates both intracellular and extracellular signaling pathways. Exogenously administered Humanin both in vitro and in vivo rapidly increases AKT-1 phosphorylation and activates the PI3K (Phosphoinositide 3-Kinase)/AKT pathway; AKT-1 is the AKT Serine/Threonine Kinase-1 protein that plays a role in cell motility, metabolism, and proliferation [47]. Moreover, intraperitoneal injection of Humanin in vivo elevates endothelial NOS (Nitric Oxide Synthase) phosphorylation and also increases the phosphorylation of AMPK (5 Adenosine Monophosphate-Activated Protein Kinase), a protein which contributes to cellular energy homeostasis [25]. The signaling pathway mediated by Humanin involves its interaction with various molecular entities, including protein kinases, integral membrane receptors, and transcription regulators. In neuronal cell lines, Humanin interacts with and activates the GP130 receptor and mediates its effects via its canonical AKT, ERK1/2 (Extracellular Signal-Regulated Kinase 1/2), and STAT3 signaling cascades. Humanin acts as a primary agonist for the GP130 receptor in neuronal cell lines in vitro, and Humanin treatment transiently activates GP130, thereby mediating cellular protective effects, such as anti-apoptosis, enhanced insulin sensitivity, and protection from hypoxic and ischemic stressors [48].

### *4.5. Preserves Endothelial Function in Atherosclerosis*

Oh et al. demonstrated that sustained administration of Humanin G (a more potent analog of Humanin) to ApoE-knockout mice inhibited the progression of atherosclerotic plaques and preserved endothelial function [49].

### *4.6. Prevents Vascular Remodeling and Inflammation*

Exogenously added Humanin attenuated angiogenesis, inflammation, apoptosis, and microvascular remodeling in an ApoE-deficient mouse model of atherosclerosis [23].

### *4.7. Cytoprotective Against LDL-Induced Oxidative Stress*

Bachar et al. demonstrated that Humanin is expressed in the endothelial cell layer of human blood vessels. In human endothelial cells in vitro, exogenous supplementation of Humanin attenuated oxidized (Ox)-LDL-induced ROS generation and apoptotic cell death by 50% and reduced the levels of cellular ceramide, which is a lipid second messenger that initiates apoptotic signaling cascades. Therefore, Humanin renders protection against Ox-LDL-mediated oxidative stress and is cytoprotective in human vasculature [50].

### *4.8. Protects Germ Cells*/*Leukocytes—Reduces Cancer Metastases*

A recent study by Jia et al. provided evidence of the role of Humanin in maintaining germ cell homeostasis. Substantive Humanin-mediated protection against chemotherapy-induced male germ cell apoptosis both in vivo and ex vivo was observed. The study also demonstrated that this anti-apoptotic effect is primarily mediated via STAT3 and BAX signaling [51].

### *4.9. Germ Cell Apoptosis by Chemo Drugs*

Another similar study by Lue et al. demonstrated that Humanin G prevents cyclophosphamideinduced toxicity and death of male germ cells and leukocytes. In this study, HNG protected normal cells against stress and suppressed cancer metastases [52].

### *4.10. Cytoprotection in Carotid Atherosclerotic Plaques*

In addition, it has been reported that Humanin is present in carotid atherosclerotic plaques and higher expression of Humanin in symptomatic patients compared to asymptomatic patients could be a part of a stress-response defense mechanism to delay the progression of the disease [53].

### **5. SHLPs (Small Humanin-Like Peptides)**

The 16S rRNA region of the mitochondrial DNA also codes for another category of MDPs called Small Humanin-Like Peptides (SHLPs), which include SHLP1, SHLP2, SHLP3, SHLP4, SHLP5, and SHLP6. SHLPs are 24–38 amino acids long and each SHLP may differentially regulate mitochondrial and cellular health and functions. Extensive characterization of SHLP2, SHLP3, and SHLP6 has been detailed in recent literature but the specific biological functions of the SHLPs are still being studied.
