*4.1. Glycyrrhizin (GA)*

GA is a triterpene glycoconjugate naturally extracted from licorice root (*Glycyrrhiza glabra*). It is composed of two molecules of glucuronic acid and glycyrrhetinic acid aglycone [96]. This molecule inhibits the chemotactic and pathogenic functions of HMGB1 by binding directly the A and B boxes [97]. GA shows a wide range of e ffects such as antibacterial, hepatoprotective, antiproliferative, antiallergic, and antiviral [98].

As a result of recent studies on ARPE cells, it has been demonstrated that HMGB1 is connected to the increase in angiogenesis and fibrosis during the course of DR [94]. Oral administration of GA in diabetic mice strongly inhibited HMGB1 concentration in retinas. This result led to a reduction in vascular and neuronal damage related to DR. The anti-inflammatory e ffects of GA were mediated by the inhibition of TNF-<sup>κ</sup>, IL-1β, and the cleavage of caspase-3 in retinal EC. GA, through the inhibition of HMGB1, reduces ROS concentrations and blood circulating glucose [99]. In another work, GA reduced TLR-4 concentrations and ischemia-reperfusion damage as well as increasing the expression of insulin receptors, partially preserving the anatomical integrity of the retina [100]. In a recent study, Liu et al. demonstrated that exchange protein for cAMP1, an inflammatory molecule involved in leukostasis, acts in synergy with GA. The combination of these two proteins strongly inhibits HMGB1 through the activation of SIRT1. SIRT1 deacetylates HMGB1, exerting a protective role in the diabetic retina [101]. GA also suppresses the proangiogenic e ffects of HMGB1 as it blocks AGE-induced upregulation of VEGF [81]. Abu El-Asrar and Mohammad's workgroup demonstrated, in a diabetic murine model, that GA can inhibit HMGB1's cytokine-like activities. Specifically, oral GA determines a reduction in HIF-1 α, transcription factor Egr-1, TYK2, CXCL12, and CXCR4 [93]. Moreover, the same authors demonstrated that GA can inhibit the upregulation of STAT-3 induced by HMGB1 and its translocation in retinal Müller cells [68], upregulate BDNF expression in experimental mice [90], attenuate the expression of NOX2, caspase-3, and PARP-1 in the retinas as well as lowering the concentrations of ROS [69] and cleave caspase-3 glutamate and downregulating neurodegeneration mediators and markers in murine retinas [92] and attenuating the expression of retinal ICAM-1 [84]; lastly, it inhibits HMGB1 mediated activation of NF-κB. [66]. GA in association with resveratrol shows the ability to replenish retinal SIRT1 expression [72].

All this evidence points in the direction of the potential use of GA in the prevention of DR and its complications. It is worth mentioning that, in human studies on male patients a ffected by chronic hepatitis and type 2 diabetes, the administration of GA reduced serum testosterone aggravating insulin resistance, atherosclerosis, and sexual dysfunctions [102]. Further studies, especially on human subjects, are needed in order to confirm the pathways and molecules involved and their e fficacy and safety.

#### *4.2. Small Interfering RNAs*/*Short Hairpin RNA (siRNA*/*shRNA)*

Small interfering RNAs (siRNAs) are a class of double-strand RNA usually constituted by 21–25 nucleotides that are gaining importance as therapeutic tools in numerous diseases. SiRNAs are capable of selectively binding specific genomic sequences, silencing them and therefore inhibiting the protein expression [103,104].

SiRNA HMGB1 transfection can repress HMGB1 RNA overexpression, determining the suppression of TLR-4 and NF-κB mRNA in RGCs. The downregulation of these inflammatory pathways can promote the survival and growth rates of RGCs [76]. A similar study conducted by Jiang and Chen confirmed these results in both in vivo and in vitro models. HMGB1 suppression, mediated by intravitreal injections of siRNA, is capable, in diabetic rats, of reducing retinal apoptosis rates as well as improving retinal function. In HRECs exposed to high glucose concentrations, siRNA HMGB1 improved cell viability and reduced the oxidative damage lowering ROS production [105]. The same study group demonstrated the protective role of HMGB1 inhibition in murine DR models. Retinal cells isolated from 8-year-old rats were incubated with a recombinant lentivirus containing short hairpin RNA (shRNA) for HMGB1. Through this mechanism, the authors obtained the silencing of HMGB1 gene expression. The results showed the downregulation of both MAPK and NF-κB, contributing to the reduction of inflammation, cell death, and BRB breakdown [83].

#### *4.3. Polygonum Cuspidatum (PCE)*

*P. cuspidatum*, also known as "Hojang-geun" in Korea, is a commonly employed herbal medicine in East Asia. The plant shows anti-inflammatory and anti-diabetic e ffects [106]. Recent works have explored the potential role, as a preventive treatment, of *P. cuspidatum* extract in diabetic nephropathy [107]. PCE is rich in resveratrol, polidatyn, and emodin compounds, with strong anti-inflammatory properties [108]. Sohn et al. suggested a beneficial e ffect of the ethanol extract of the root in a DR murine model. It prevents diabetic-induced retinal vascular hyperpermeability, attenuating the HMGB1 signaling pathway through the downregulation of the RAGE-mediated activation of NF-κB. It directly blocks the binding of HMGB1 to RAGE, thus preventing retinal vascular inflammation. Moreover, fluorescein angiography demonstrated that PCE markedly inhibits fluorescein leakage, suggesting that it may prevent the breakdown of the BRB. PCE reduces the expression of HMGB1 in diabetic rat retinal tissue and inhibits the binding of NF-κB to the RAGE promoter, with considerable anti-inflammatory activity. It is worth mentioning that the oral administration of PCE showed no positive e ffects on glycemic and body weight control in the murine model [109].
