Puerarin as potential treatment in diabetic retinopathy

Introduction Diabetes mellitus is a progressive metabolic disorder characterized by high levels of blood glucose. Based on the report of International Diabetes Federation (IDF), the number of patients with diabetes is estimated to be doubled from 415 million in 2015 to 642 million in 2040 (1). Despite the high prevalence and rising trend, the microand macrovascular complications of diabetes are the main cause of morbidity and mortality, reduction in life expectancy, and high healthcare expenditures, particularly in diabetic elderly patients (2). Diabetic retinopathy (DR) is one of the most prevalent complications of diabetes and the leading cause of visual loss among working-aged adults around the world (3). In a study, 92.6 million (34.6%) patients with diabetes suffered from DR, of which 28.4 million patients afflicted with serious vision impairment (4). Several factors, including type and duration of diabetes, glycemic control and insulin resistance status, and the presence of risk factors of DR such as hypertension, smoking, and dyslipidemia are involved in the severity of DR (5). The most effective strategies to delay the onset and prevent the progression of DR are tight glycemic control and treatment of comorbidities of diabetes including high blood pressure and dyslipidemia (6). Laser photocoagulation and vitrectomy are now the only approved treatments of late stages of DR (7). Recently, development of various pharmacological agents has remarkably improved the management of vision loss and retinal dysfunction (8). Intravitreal pharmacotherapy with anti-angiogenic and anti-inflammatory agents is the most commonly used protocol for both prevention and treatment of established DR (9). Moreover, several lines of studies are investigating and developing different classes of chemical constituents to manage DR. Natural remedies as invaluable and available compounds also have gotten attention of the researchers. Puerarin is a polyphenolic compound isolated from http://www.herbmedpharmacol.com doi: 10.34172/jhp.2020.14


Introduction
Diabetes mellitus is a progressive metabolic disorder characterized by high levels of blood glucose. Based on the report of International Diabetes Federation (IDF), the number of patients with diabetes is estimated to be doubled from 415 million in 2015 to 642 million in 2040 (1). Despite the high prevalence and rising trend, the micro-and macrovascular complications of diabetes are the main cause of morbidity and mortality, reduction in life expectancy, and high healthcare expenditures, particularly in diabetic elderly patients (2).
Diabetic retinopathy (DR) is one of the most prevalent complications of diabetes and the leading cause of visual loss among working-aged adults around the world (3). In a study, 92.6 million (34.6%) patients with diabetes suffered from DR, of which 28.4 million patients afflicted with serious vision impairment (4). Several factors, including type and duration of diabetes, glycemic control and insulin resistance status, and the presence of risk factors of DR such as hypertension, smoking, and dyslipidemia are involved in the severity of DR (5).
The most effective strategies to delay the onset and prevent the progression of DR are tight glycemic control and treatment of comorbidities of diabetes including high blood pressure and dyslipidemia (6). Laser photocoagulation and vitrectomy are now the only approved treatments of late stages of DR (7). Recently, development of various pharmacological agents has remarkably improved the management of vision loss and retinal dysfunction (8). Intravitreal pharmacotherapy with anti-angiogenic and anti-inflammatory agents is the most commonly used protocol for both prevention and treatment of established DR (9). Moreover, several lines of studies are investigating and developing different classes of chemical constituents to manage DR. Natural remedies as invaluable and available compounds also have gotten attention of the researchers.
Puerarin is a polyphenolic compound isolated from various plants and herbs and intensively investigated for several beneficial activities. It is widely used as adjuvant agent in the treatment of neurodegenerative diseases, diabetes and its complications, cardiovascular and cerebrovascular diseases, and cancer (10,11). A number of studies have shown this compound could prevent, and improve retinopathy in diabetes. In the present review, the beneficial impacts of puerarin in the treatment of DR were screened and the underlying mechanisms behind these pharmacological activities completely discussed.

Pathophysiology of DR
Although the exact pathogenesis of DR is not thoroughly understood, chronic hyperglycemia is seemed to be the primarily cause of this disease (12). There are four known molecular mechanism describing how hyperglycemia results in DR. The polyol pathway flux increases under hyperglycemic condition. Aldose reductase reduces intracellular glucose to sorbitol, and aldehydes to inactive alcohols with depletion of nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione. Subsequently, sorbitol dehydrogenase oxidizes sorbitol to fructose with consumption of nicotinamide adenine dinucleotide (NAD + ) (13). Moreover, intracellular production of advanced glycation end-products (AGEs) increases under hyperglycemic condition. Non-enzymatic modifications of intracellular and matrix proteins alter various cellular functions, and cause abnormal interactions between several matrix proteins and integrins. Changes of plasma proteins produce ligands which bind to receptors of AGE (RAGEs), and result in generation of intracellular reactive oxygen species (ROS), and changes in expression of several gens (14). Intracellular hyperglycemia also increases diacylglycerol content, and activates protein kinase C, which has numerous pathogenic effects, especially activation of nuclear factor kappa light chain enhancer of activated B cells (NFκB) and NADPH oxidases (15). Furthermore, hyperglycemia-induced activation of the hexosamine pathway leads to several alternations in both gene expression and protein function (16).
A body of evidence has demonstrated hyperglycemiainduced oxidative stress and inflammation along with apoptosis are the main reasons responsible for overexpression of retinal growth factors (particularly vascular endothelial growth factor, VEGF), retinal hemodynamic changes, and impairment in neurotrophic factor receptors and their signaling pathway, which damage to retinal vessels, neurons, and glial cells (3,17,18). These pathogenic mechanisms involve in retinal microvasculopathy as well as early neuropathy, and lead to retinopathy and vision impairment.

Puerarin
Puerarin as one of the most famous isoflavone was isolated from Pueraria lobata in the 1950s for the first time. The polyphenolic structure of puerarin is demonstrated in Figure 1. Several line of studies have intensively investigated the pharmacological properties of this compound. There are some commercially dosage forms of puerarin for oral and injection applications, and currently used as an adjuvant therapy in the management of cancer (11), neurodegerative diseases (19) including Alzheimer's and Parkinson's diseases, vascular diseases (20), liver injuries (21), and osteoporosis (22). Injection form of puerarin has been approved by State Food and Drug Administration of China (SFDA) for the treatment of myocardial infarction as well as angina pectoris. Documented evidences have also demonstrated puerarin has a protective role against diabetes mellitus (23) and its complications, specially DR.

Diabetic retinopathy and puerarin
The protective effects of puerarin have been investigated against DR in several pre-clinical and clinical studies. These beneficial impacts attributed to the abilities of this constituent to inhibit retinal oxidative stress, inflammation, apoptosis, vasculopathy, and neuropathy in hyperglycemic milieu. The effects of puerarin on in vitro and animal models of DR are summarized in Tables 1 and  2, respectively.

Antioxidant effects
Several studies have shown that puerarin is able to act as a strong antioxidant factor, which exerts its effect via several pathways. Puerarin protects cells from oxidative-induced damage via induction of activity of antioxidant enzymes of glutathione S-transferase (38), superoxide dismutase (SOD), and catalase (39). Furthermore, puerarin acts its protective effect via activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which is a potent regulator for cellular defense system against toxic agents. Moreover, it has been shown that puerarin prevents complications of diabetes via attenuation of mitochondria derived ROS, especially by inhibition of NADPH oxidase (40,41). On the other hand, puerarin, a phytoestrogen, is assumed to exert its antioxidant activities through induction of antioxidant/electrophile response element (ARE/EpRE)-mediated gene expression by activation of Nrf2-related factor 2-Keap 1 signaling pathway (42).
AGE is one of the sources of ROS under hyperglycemic condition, which plays a critical role in the pathogenesis of DR (43). AGE-mediated damages are exerted via interaction of AGEs to the RAGEs, which activates ROS producing enzyme of NAPDH oxidases, and consequently, increased intracellular ROS formation (44). Several studies have reported that the increased production of ROS in retinal tissue of animal models (26) as well as in vitro models (24)(25)(26)28) of retinopathy attenuated in the presence of puerarin. Moreover, puerarin caused a decrease in the production of retinal AGE (30,34) along with a reduction in the expression and protein level of RAGE (33,34) in animal models of DR. Puerarin also diminished the NADPH oxidase activity of bovine retinal pericyte cells (BRPCs) in AGE-induced retinopathy model (28).
Puerarin decreased the expression of retinal inducible nitric oxide synthase (iNOS) in STZ-induced diabetic rats (32,36), and inhibited over production of nitric oxide (NO) and peroxynitrite (26). The protective effects of puerarin is mediated via inhibition of over expression of iNOS and peroxynitrite generation mediated through cell surface death receptor/cell surface death receptor ligand (Fas/FasL) signal pathway (32,36). Moreover, the

Anti-inflammatory effects
As it was mentioned before, inflammatory changes have been known as important contributors in the pathogenesis of DR. So, attenuation of inflammation could be invaluable in preventing retinal alterations in diabetic condition. Puerarin is able to act as an effective immunomodulator, and affect a wide range of inflammatory mediators including interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and iNOS (45). It has been shown that antiinflammatory activity of puerarin is mediated through alteration of several inflammatory associated signaling pathways, including extracellular signal-regulated kinase 1and 2 (ERK), and c-Jun N-terminal kinase (JNK) (46), NF-κB signaling cascade (47), insulin receptor substrate-1 (48), phosphoinositide 3-kinases (PI3Ks) and antioxidant response element-luciferase and translocation of Nrf2 (49), in various inflammatory models. The anti-inflammatory activity of puerarin against DR has been examined in several studies. This compound inhibited IL-1β-mediated leukostasis in rat retinal capillary endothelial cells (TR-iBRB2) via a decrease in expressions of vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Therefore, it is able to inhibit adhesion of leukocytes to the studied cells, and prevent consequent inflammatory  events. Puerarin also inhibited inflammatory response by attenuating the activity and decreasing the level of NF-κB in retinal tissue of STZ-induced diabetic rats (35) and AGE-induced retinopathy in BRPC (28).
In spite of the current reports, it seems that the antiinflammatory effect of puerarin, especially in DR, needs further study. Future works should be focused on further clarifying the possible underlying mechanisms of action and finding more effective potential targets for the therapy of DR.
Vasculoprotective activities As mentioned, damage to retinal microvasculature in hyperglycemic condition has a crucial role in pathogenesis of DR. VEGF as a key factor has an underlying role in retinal neovascularization and proliferative DR. Puerarin attenuated the expression of retinal VEGF in STZ-induced diabetic rats (33,34). In addition to the downregulation of VEGF, puerarin decreases the expression of retinal hypoxia inducible factor 1 (HIF-1α) and modulator of VEGF expression in hypoxic condition, in diabetic rats (37). Hyperglycemia-induced over expression and function of iNOS can possess endothelial dysfunction in retinal tissue, which has a main role in the pathophysiology of DR. Several studies have reported that puerarin decreased the expression of iNOS in in vitro (26,29) and animal models (32,36) of DR. It has been mentioned that puerarin potentially is able to prevent blood-retinal barrier breakage.
Neuroprotective activities Recent studies have reported that retinal neurodegeneration as well as retinal vasculopathy are involved in the pathogenesis of DR, particularly in the early stages of diabetes (12). Puerarin prevented retinal bipolar cell death in animal model of STZ-induced retinopathy (37), and RGC loss in NMDA-induced retinopathy (26). This compound also improved the decreased thickness of outer nuclear layer (ONL) in STZ-induced diabetic rats (34). Puerarin diminished glutamate-induced neurotoxicity in Y-79 cells, and the neuroprotective effects might be attributed to the inactivation of mitochondrial-dependent signaling pathway in the presence of glutamate. Puerarin inhibited CaMKII-dependent apoptosis signal-regulating kinase 1 (ASK-1)/JNK/p38 MAPK signaling pathway (25). The neuroprotective properties of this compound also studied in electroretinography of STZ-induced diabetic rats. Puerarin restored b-wave amplitude and improved retinal function (31,35).
Human studies Three studies have examined the therapeutic effects of puerarin in patients with DR (50)(51)(52). Different parameters of retinal hemodynamic and hemorheology were assessed using color Doppler ultrasonography method in diabetic patients. The obtained results demonstrated that puerarin improved the peak systolic and end diastolic velocity of central retinal artery as well as central retinal vein reflux velocity (51,52). Moreover, puerarin restored naked eye vision and attenuated clinical symptoms of DR. A brief summary of clinical studies evaluating the role of puerarin in DR is presented in Table 3. It will be useful to evaluate the safety and tolerability of puerarin rather than its effectiveness. Hence, high-quality clinical trials with larger population size and longer period of intervention should be conducted to evaluate the impacts of this compound on DR.

Conclusion
Puerarin attenuates retinal neovascularization and neurodegeneration in diabetes. The beneficial effects are attributed to the antioxidant, anti-inflammatory, and antiapoptotic properties of the compound. Puerarin might be a potential adjuvant agent for the prevention and treatment of DR. however, adequate and high-quality clinical trials with larger population size and longer duration are necessary to show its effectiveness and safety.