*4.3. EGCG*

Polyphenols such as epigallocatechin-3-gallate (EGCG) derived from green tea are showing potential regenerative effects. In vitro, C2C12 myoblasts exposed to polyphenolic EGCG showed a higher number of myotubes with increasing length [85,86]. EGCG activates the transcriptional coactivator TAZ that in turn increases myogenin, myoblasts fusion, and myotubes formation [85]. TAZ is related to tissue homeostasis, regeneration, organ development, and myogenic differentiation that occurs through the stimulation of the myogenic differentiation factor (MyoD) [85,87]. Similarly, EGCG stimulates miRNA-486- 5p expression and reduces myostatin promoting C2C12 myoblasts differentiation [86]. Myostatin is a member of the transcription growth factor β (TGFβ) family that inhibits

satellite cell proliferation, myoblasts differentiation, and in vivo induces fibrosis in severely injured muscles [88,89].

In a study using senescent mice was provided an enriched-EGCG diet for up to eight weeks [86]. Their results showed a gradual increasing of muscle mass in this group than in the group without this diet [86]. A similar result was found in a mice model of dystrophy that showed improvements in force production in the treated mice for up to eight weeks with EGCG than in the untreated group [90]. In the same study, the dystrophic diaphragm and soleus showed a higher area of skeletal muscle and lower area of connective tissue in time [90]. Aged rats were used in a hindlimb muscle atrophy disuse model [91]. The treatment with EGCG induced reduction of pro-apoptotic pathways, satellite cell proliferation, cell differentiation, and force production in the treated group [91]. In this group, oxidative markers were lower while SOD antioxidant markers were higher [91]. In a different study, the crushed nerve injury in the hindlimbs of rats was treated with EGCG intraperitoneally for up to 8 weeks [92]. There were increasing myelin sheath thickness, nociceptive recovery, hindlimb reflex, and posture improvements such as standing on the injured limb in the treated groups [92]. This study did not describe the functional or histological analysis of hindlimb skeletal muscles. However, the recovery in posture and coordinated movements indicate potential recovery in the injured muscles. All this evidence suggests the activation of regenerative mechanisms in nerves and skeletal muscles induced by exogenous antioxidant effects.

Another molecular mechanism involved in skeletal muscle regeneration induced by EGCG is similar to the effects observed in insulin-like growth factor (IGF-1). In vitro, IGF-1 enhances myotube formation in aligned scaffolds showing increasing length and higher number of nuclei per myotube [93]. In vivo, IGF-1 delivered in aligned scaffolds implanted in large excisional wounds showed myofiber formation and lower areas of collagen in time [93]. In recent in vitro studies, skeletal muscle fibers isolated from mice were stimulated to express Forehead box (Foxo)-O1 and Foxo-O3 [94,95]. These proteins are implicated in muscle atrophy mainly by increasing protein degradation via E3 ubiquitin ligase expression [94,95]. A decreased nuclear Foxo expression was observed in muscle fibers exposed to IGF-1 and EGCG but not in the untreated cells [95]. The addition of ROS such as H2O2 to the EGCG group cells did not increase nuclear Foxo activity [95]. All this indicates that EGCG might promote additional protective effects in skeletal muscle fibers comparable to those that occur upon IGF-1 stimulation.

In situ, in a model of severe nerve and muscle radiation injury in the rat hindlimb was delivered EGCG in a biodegradable synthetic hydrogel scaffold [96]. It was observed three months later increased antioxidant markers Nrf2 and MnSOD alongside reduced nNOS and TNFα [96]. The area of the regenerated nerve was higher in the EGCG-hydrogel group than in the untreated group. The myelin sheath thickness and the number and diameter of axons were also higher in the treated groups with EGCG-hydrogel [96]. In the same study, the area of muscle fibers in the fibrotic muscles due to radiation exposition showed higher recovery in the EGCG-hydrogel group than in the untreated group. Similarly, strength and muscle mass were higher in the treated group [96]. The in vitro component in this study included the effects of the scaffold EGCG-loaded using Schwann cell and skeletal muscle fibers isolated from rats [96]. Their results one week after culture showed higher proliferative ability and differentiation of both cell types in EGCG-hydrogel than in control cultures [96]. All this preclinical evidence indicates that EGCG stimulates nerve regeneration and potential recovery of atrophic muscles. All this is promising for therapeutic approaches in nerve and skeletal muscle regeneration.

In a clinical study, 60 years-old patients were treated with EGCG for 12 weeks [97]. Their main results include increases in antioxidant activity, reduction of myostatin, and grip strength [97]. The autoimmune and inflammatory effects seen in multiple sclerosis disease are mostly demyelination and skeletal muscle damage [98]. A daily dose of 600mg of EGCG was provided for up to 12 weeks to patients upon treatment for multiple sclerosis [98]. After moderate exercise, improvements in muscle metabolism such as lactate

reduction, and stable carbohydrate oxidation were found over time [98]. Further analysis beyond this study may include specific neural and muscular parameters for regeneration in these patients receiving EGCG along with their treatment. This clinical evidence shows regenerative effects of EGCG in the recovery of severely impaired skeletal muscle due to demyelination. In summary, EGCG seems to stimulate molecular mechanisms for satellite cell differentiation, skeletal muscle growth, and regeneration.

### *4.4. Vitamins and Other Elements*

Regarding vitamins and other remyelinating components, vitamin E promotes myelin maintenance and prevents cell damage as shown in mice [99]. The lack of this vitamin causes axonopathy and demyelination [99]. The administration of vitamin E and vitamin D3 in a rat model of multiple sclerosis showed antioxidant effects, reduced apoptosis, and increased remyelination [100]. The treatment of the sciatic nerve injury in rats with vitamin E mixed with pyrroloquinoline-quinone, a small molecule with antioxidant effects, showed improvement in nerve function, increased muscle mass, and motor functional recovery [101]. Vitamin C, or ascorbic acid, is also involved in peripheral nerve development and maintenance for collagen synthesis and lipid protection showing antioxidant effects [17].

Selenium is a trace element considered an essential micronutrient for eukaryotes and procaryotes through the function of selenoproteins (Se1P). Knockout mice for this protein showed problems in learning during training and impaired motor coordination [102]. Se1P incorporates selenium in the form of selenocysteine and selenium-methionine residues [103]. In the CNS, selenium acts as a cofactor for glutathione peroxidase types I–IV and VI [104].


**Table 1.** Exogenous antioxidant effects on muscle and nerve regeneration.

Selenium is also important in the function of SelP, a selenium-rich protein, present in glial cells, the choroid plexus, and cerebral spinal fluid [104]. SELENOP1 is a metal binding protein with antioxidant functions able to regulate the concentration of tau phosphorylated protein and synaptic Zn<sup>2</sup> in a mice model [105]. Finally, 18β-glycyrrhetinic acid is a compound found in licorice roots shown to suppress the proinflammatory chemokines CCL2, CCL3, CCL5, CXCL10, and CCL20 in mice models of encephalomyelitis [106]. This

121

study showed microglial inhibition and increased remyelination possibly via the expression of brain-derived neurotrophic factor (BDNF) [106]. All these vitamins and elements are showing potential remyelinating effects worthy of deeper research. In general, the evidence in this review is showing promising effects in remyelination and muscle regeneration mostly induced by exogenous antioxidants.

Further strategies in sync with pharmacological treatment and antioxidant approaches include physical activity. Exercise is another therapeutical intervention to reduce oxidative mechanisms following demyelination to recover muscle mass and function [107,108]. The genetic approach in mice models also aims to stimulate and accelerate myelin production reducing inflammatory responses [64]. One of the mechanisms of accelerated remyelination is the activation of peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC1a) that increases during exercise in mice [109]. It seems that accelerating remyelination after autoimmune inflammation protects the axon structure and prevents axonal loss in the central nervous system.

To last, a growing worldwide disorder triggering neurodegeneration is obesity [35,110]. This condition increases nerve damage and shows impaired nerve regeneration also in absence of diabetes [35,110]. In obese rats, the recovery of mechanical injuries of the sciatic nerve showed a lower number of myelinated axons and thinner myelin sheath than the normal weight group [111]. Similarly, hyperlipidemia, or high levels of lipids in the blood, constitutes a risk that may induce neuropathy and peripheral nerve dysfunction [112]. Lowering weight and preventing hyperlipidemia may reduce the risk and peripheral neuropathy effects [113]. Obesity may also cause muscle damage and impair skeletal muscle regeneration [114]. Satellite cells from limb muscles in obese mice showed reduced activity of AMP-activated protein kinase α [114]. This enzyme stimulates myogenin expression and myoblasts fusion for its inhibition decreases the regenerative ability in skeletal muscles [114]. Oxidative stress and exacerbation of multiple inflammatory responses in obesity induce the inhibition of AMP-activated protein kinase α [114,115]. Then, based on the evidence in this review, exogenous antioxidants such as flavonoids, EGCG, and vitamin E may reduce oxidative damage and inflammation improving the regenerative ability of nerves and skeletal muscles.

To conclude, autoimmune and inflammatory mechanisms cause demyelination and nerve damage that harms skeletal muscles causing myopathy-like destruction. Oligodendrocytes and Schwann cells show adaptive responses upon injury and incomplete remyelination. Approaching mechanisms such as Nrf2, ORMDL, proteasomes, and others, might improve remyelinating therapies. Additional targets with potential pharmacological interest include the metabolic regulation of ceramides and their association with inflammatory mechanisms. Preclinical evidence using antioxidants such as curcumin, EGCG, and other exogenous antioxidants showed regenerative effects. These effects include stimulation of oligodendrocyte and Schwann cell differentiation, increased myelin production, reduction of oxidative stress, increased endogenous antioxidant activity, recovery of the neuromuscular junctions, and skeletal muscle functional recovery. Then, inducing simultaneous regeneration of nerves, skeletal muscle, and neuromuscular junctions along with current approaches will enhance the overall recovery discussed in this review.

**Funding:** The APC was funded by the Central Office of Research, Universidad del Tolima, gran<sup>t</sup> number 20520.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
