**4. Discussion**

In our previous studies, we examined the effects of herbal extracts in inhibiting AGE formation (CPA1-1 (1:1) IC50 = 9.94 ± 0.20 μg/mL; CPA2-1 (1:2) IC50 = 7.54 ± 0.18 μg/mL; CPA3-1 (1:4) IC50 = 8.16 ± 0.23 μg/mL; CPA4-1 (1:8) IC50 = 6.84 ± 0.08 μg/mL) and we showed potential therapeutic targets of DR in animal and in vitro models. In this study, we investigated whether CPA4-1, a herbal combination, inhibits AGE formation and breaks the cross-linking of AGE with collagen in vitro. We tested whether CPA4-1 affects the expression of occludin in the retina and whether this effect is correlated with inhibition of BRB breakage. Our findings showed that CPA4-1 significantly prevented retinal vascular leakage and retinal acellular capillary formation. These results indicated that CPA4-1 attenuated the downregulation of tight junction protein occludin in the retinas of db/db mice. The tight junction acts as a semipermeable barrier for paracellular transports of solutes, ions, water, and cells. Occludin is the first identified component of the tight junction strand, a transmembrane protein of approximately 60 kDa [24]. In diabetic patients, retinal occludin protein expression is selectively decreased, and BRB permeability is increased [25]. Decreased expression of tight junction proteins is important in diabetic patients, as it causes increased vascular permeability, vasodilation, and edema. As CPA4-1 was shown to increase the expression of occludin in the retina of db/db mice in this study, CPA4-1 might serve as an inhibitor of AGE formation to protect against DR.

Fasting blood glucose and HbA1c levels are important factors in diabetes. HbA1c is monitored in the long-term follow-up of patients with DR, and diabetic patients are primarily treated with insulin to lower blood glucose levels. Fenofibrate decreased blood glucose level and HbA1c level in the db/db mice group (Table 3). Fenofibrate treatment induces lowing of blood glucose levels and HbA1c by reducing adiposity, thus improving peripheral insulin action [26]. Previous studies have suggested that that fenofibrate exerts robust protective e ffects against DR in type 2 diabetic patients and type 1 diabetic animal models [27,28]. The potential therapeutic mechanisms of fenofibrate on retinal microvascular dysfunctions induced by diabetes include the restoration of VEGF and the reduction of oxidative stress in diabetic rats [29]. As CPA4-1 did not alter blood glucose level and HbA1c of db/db mice compared with those of the db/db mice group, CPA4-1 might act as a supplement for the prevention of BRB breakage and retinal acellular capillary formation, without altering blood glucose level or HbA1c of diabetic patients.

Herbal medicine has been used to treat diabetes and diabetic complications, including DR, for thousands of years worldwide. Many researchers have tried to provide evidence of the therapeutic effectiveness and mechanisms of herbal medicine. *Panax notoginseng* (Araliaceae) has been reported to exert antidiabetic activities, and it contains saponins, such as ginsenoside Re 14, ginsenoside Rd, ginsenoside Rg1, ginsenoside Rb1, and notoginsenoside R1. These saponins have also been reported to exert antioxidant action [30]. As free radical damage is critically involved in the pathophysiology of DR, *P. notoginseng* saponins can be used in the treatment of DR owing to their beneficial e ffects. *Pueraria lobata* (Fabaceae) is one of the most used herbs in traditional Korean medicine, and it contains puerarin, genistein, and daidzein. Puerarin directly inhibits the formation of AGEs in vitro [31]. CPA4-1 is formulated at the optimum conditions to inhibit AGE formation. Cinnamic acid from Cinnamomi Ramulus promotes the managemen<sup>t</sup> of diabetes and its complications [32]. Cinnamic acid and its derivatives stimulate insulin and hepatic gluconeogenesis, enhance glucose uptake, and inhibit AGE formation [33,34]. Paeoniae Radix extract contains 5% paeoniflorin, the most important component that suppresses high glucose-induced retinal microglial matrix metalloproteinase 9 (MMP-9) expression and inflammation by reducing capillary permeability [35]. Paeoniflorin also exerts a neuroprotective effect on inflammation and apoptosis in Alzheimer's disease and regulates hepatic cholesterol synthesis and metabolism by reducing oxidative stress [36,37].
