Association Between Silymarin Use and Age-related Macular Degeneration in Patients With Chronic Hepatic Diseases: a Population-based Retrospective Cohort Study


 Silymarin is a bioflavonoid compound mostly used in patients with chronic hepatic diseases (CHDs). It has been shown to inhibit retinal angiogenesis in in vitro and in vivo animal studies. Age-related macular degeneration (AMD) is a leading cause of vision impairment worldwide, and no definite preventive measures for AMD exist at present. Through this study, we aimed to investigate whether the use of silymarin affects the risk of AMD in patients with CHDs. Cox proportional hazard models were used to assess the hazard ratio (HR) of AMD in this population-based retrospective cohort study. The CHDs was associated with a higher risk of AMD (adjusted hazard ratio (aHR): 1.33, 95% CI 1.22–1.46), and the use of silymarin reduced the risk of AMD among patients with CHDs (aHR: 0.76, 95% CI: 0.67–0.87). A higher accumulative dose (defined daily dose [DDD] < 631 mg) of silymarin was associated with a lower risk of AMD, but the difference was nonsignificant. In conclusion, CHDs were associated with a higher incidence of AMD, and the use of silymarin was associated with a reduced risk of AMD in patients with CHDs. However, a higher accumulative DDD of silymarin did not reduce the risk of AMD.


Introduction
Age-related macular degeneration (AMD) is a disease that affects the macular region of the retina, causing the progressive loss of central vision. It is currently the third leading cause of vision impairment worldwide, and the number of individuals with AMD is expected to reach approximately 200 million by 2020, posing a global health burden 1,2 . AMD is a disease of the aging eye, with multiple factors including genetics and environmental factors affecting its course 1,3 . Among these factors, oxidative stress had attracted much interest. Large randomized controlled trials have shown bene cial effects of the use of high-dose oral antioxidant vitamins in slowing the progress of AMD, leading to their inclusion in the treatment of AMD by some authorities [4][5][6] . Other nutrients with antioxidant effects, such as anthocyanins and selenium, were also investigated 7 .
Silymarin, a bio avonoid, is a natural compound extracted from Silybum marianum, or milk thistle 8 . Silymarin has been used as a traditional Chinese herbal medicine for more than 2,000 years, mostly in patients with chronic hepatic diseases (CHDs) such as viral hepatitis, nonalcoholic fatty liver disease, and cirrhosis 8 . It was found to have multiple potential pharmacological properties, such as antioxidant, antiin ammatory, and hepatoprotective effects 9 . An in vitro study found that silibinin, the main bioactive component of silymarin, inhibited the secretion of vascular endothelial growth factor (VEGF) from retinal pigment epithelium cells 10 . In a rat model of AMD, it inhibited retinal edema and angiogenesis caused by VEGF and hypoxia conditions 10 . However, whether these results can be extrapolated to humans is still unknown. Thus, through this study, we aimed to investigate the effect of silymarin on the risk of AMD by using a nationwide health insurance database. Because silymarin is usually used in patients with CHDs, this speci c population was analyzed.  11 . The HWDC established and veri ed the sampled database.

Study design and patient selection criteria
The study period was from 2001 to 2014, and our patient enrollment period was from 2001 to 2009. The study owchart is depicted in Figure 1. The International Classi cation of Diseases, ninth revision (ICD-9) code 571.X was used for the con rmation of CHD diagnoses, and codes 362.50, 362.51, and 362.52 were used for the diagnosis of AMD. Patients diagnosed as having CHDs twice from January 1, 2001 to December 31, 2009 were enrolled. Those aged < 40 years were excluded. The diagnosis date was de ned as the date of the rst diagnosis of CHDs, and the index date was de ned as the date of the rst recorded use of silymarin for Study Group I.
Among the patients diagnosed as having CHDs, Study Group I included patients with silymarin use and excluded patients who had used silymarin before the diagnosis date, those who had used silymarin for fewer than 90 days within 1 year after index date, those who had AMD before the diagnosis date, and those who were diagnosed as having AMD within 1 year after the index date. Among patients diagnosed as having CHDs who did not use silymarin, those diagnosed as having AMD before the diagnosis date were excluded, and those remaining were grouped into Study Group II.
From patients without CHDs or silymarin use, we randomly selected a group of patients and assigned them to the Comparison Group, which was 1:1 matched with Study Group II by age, sex, and year of diagnosis date. Because the patients in the Comparison Group were not diagnosed as having CHDs, the date used for matching was called the reference date in the Comparison Group. Patients from the Comparison Group who were diagnosed as having AMD before the reference date were excluded. This study was approved by Ethics Committee of Taipei Medical University (TMU-JIRB, No.: N201610012). Because all NHIRD data are delinked and deidenti ed, the requirement for obtaining informed consent was waived. We con rm that all methods were performed in accordance with the relevant guidelines and regulations and the study had been performed in accordance with the Declaration of Helsinki.

Risk factors and confounding factors
The potential risk factors for AMD, including hypertension, diabetes mellitus, hyperlipidemia, cerebrovascular diseases, coronary artery diseases, heart failure, peripheral vascular diseases, gastrointestinal disorders, dementia, obesity, tobacco use disorder, and alcohol abuse were identi ed in the patients. The potential confounding factors of medication use, including that of lipid-lowering agents (statin, brate), antihypertensive agents (angiotensin-converting-enzyme inhibitors, angiotensin II receptor blockers, calcium channel blockers), and aspirin, were also evaluated.

Outcome measurement
Because we aimed to investigate the long-term use of silymarin in patients with CHDs, only those who had used silymarin for more than 90 days per year were included. We evaluated the occurrence of AMD in each group as the primary outcome. For the evaluation of the dose-response as the secondary outcome, the Anatomical Therapeutic Chemical (ATC)/De ned Daily Dose (DDD) system was used 12 . Owing to lack of data regarding silymarin DDD in World Health Organization suggestions, we set its DDD to 420 mg according to the package insert 13 .

Statistical analysis
Among the different patient groups, means with standard deviations were used to describe age, and proportions were used to describe all other variables (including sex, comorbidities, and co-medication).
Student's t test and Pearson's chi-squared test were performed to evaluate the differences in all the other baseline characteristics. The Cox proportional hazards regression model was used to analyze the risk of AMD between the groups. The potential risk factors mentioned in the previous section were included in the model. Two-tailed hypothesis tests were used in all analyses, and p < 0.05 indicated statistical signi cance. SAS (version 9.4; SAS Institute, Cary, NC, USA) was used for all statistical analyses.

Baseline characteristics
The LHWD database consisted of 113,387 patients aged > 40 years and diagnosed as having CHDs at least twice from 2001 to 2009. Among them, 14,579 were enrolled into Study Group I (patients with CHDs who used silymarin) and 54,866 were enrolled into Study Group II (patients with CHDs who did not use silymarin). The Comparison Group (the 1:1 matched control group, patients without CHDs or silymarin use) consisted of 53,320 patients. The baseline patient characteristics are shown in Table 1. All characteristics other than obesity signi cantly differed between Study Groups I and II. Between Study Group II and the Comparison Group, all characteristics other than sex signi cantly differed. Risk of AMD in patients with CHDs, with or without silymarin use The risk of AMD in the different groups is shown in Table 2. Among patients with CHDs (Study Groups I and II), use of silymarin reduced the risk of AMD; the adjusted hazard ratio (aHR) was 0.76 (95% CI: 0.67-0.87). Among patients without silymarin use (Study Group II and Comparison Group), the diagnosis of CHDs was associated with a signi cantly higher risk of AMD (aHR 1.33, 95% CI 1.22-1.46). The results revealed that although patients with CHD were associated with a higher rate of AMD, silymarin use was associated with a reduction in the occurrence of AMD.  CHDs, chronic hepatic diseases; AMD, age-related macular degeneration; HR, hazard ratio; CI, con dence interval; SD, standard deviation; DDD, de ned daily dose Adjusted for age, sex, hypertension, diabetes, hyperlipidemia, cerebrovascular disease, coronary artery disease, heart failure, peripheral vascular disease, gastrointestinal disorder, dementia, obesity, tobacco use disorder, alcohol abuse, statin, brate, aspirin, calcium channel blocker, angiotensin-convertingenzyme inhibitor, and angiotensin II receptor blocker **p < 0.01, *p < 0.05

Discussion
In this study, we compared the incidence of AMD among three groups of patients by using a nationwide database: those with CHDs who used silymarin (Study Group I), those with CHDs who did not used silymarin (Study Group II), and those without CHDs or silymarin use (Comparison Group).
The study had stringent criteria; the included patients were required to have been diagnosed as having CHDs on at least two different occasions. To better evaluate the effect of silymarin on the occurrence of AMD, we included only patients with long-term silymarin use after the diagnosis of CHDs. Those who had AMD before using silymarin or within 1 year after initiating silymarin were excluded.
We found that patients with CHDs who did not use silymarin had a higher risk of AMD, with an aHR of 1.33 (95% CI, 1.22-1.46). Among patients with CHDs, use of silymarin was associated with a lower risk of AMD (aHR 0.76, 95% CI 0.67-0.87); however, no signi cant dose-dependent response was observed.
AMD is classi ed into two forms, a nonneovascular or "dry" form and a neovascular "wet" form. Anti-VEGF agents have shown to be an effective treatment for neovascular AMD, but no effective preventive measures exist 14 . Antioxidant vitamin supplements may slow the progression of AMD; however, a metaanalysis suggested that they do not prevent or delay the onset of AMD [ 15,16 ]. Various other nutrients have been tested for their possible preventive effects against AMD, and the ndings of the current study add silymarin to the list 7 .
Pretreatment of retinal pigmented epithelial cells with silibinin, the main component of silymarin, was shown to inhibit the hypoxia-induced pathway in a previous study, reducing VEGF secretion, thereby preventing hypoxia-induced angiogenesis in a rat model of AMD 10 . Other than AMD, in vitro studies have shown that silibinin can protect retinal ganglion cells from blue light-induced injury 17 and prevent hyperpermeability induced by diabetic conditions in human retinal endothelial cells 18 . Further human studies are warranted to con rm the e cacy of silibinin in preventing these eye conditions.
The risk factors of AMD include age, genetics, smoking status, and diet 1 . Liver disease was previously not considered a risk factor for AMD, but some case series have reported that retinopathy occurs in approximately 15% of patients with liver cirrhosis and 32% of patients with chronic hepatitis C 19,20 . The authors of the aforementioned studies have suggested that loss of synthetic function of the liver and hemodynamic effects of portal hypertension in patients with cirrhosis might contribute to retinopathy, and immune-mediated ischemic retinopathy might be the pathogenesis underlying chronic C hepatitisassociated retinopathy 19,21 . Pathologic angiogenesis is closely related to the brogenic progression of CHDs, with hypoxia and hypoxia-inducible factors playing a major role, and in vitro study has shown that antiangiogenic therapy is highly effective in signi cantly reducing brogenic progression 22 . We suggested that this pathologic angiogenesis also occurs in the retina, leading to a higher incidence of AMD in patients with CHDs, and can be suppressed with silymarin use.
The current study had some limitations. Because this was a retrospective study based on information from the health insurance database, the type of AMD that occurred, the type of CHD that was more related to the higher risk of AMD, and the type of AMD whose incidence was reduced by silymarin were di cult to discriminate. Further prospective trials with silymarin are warranted to answer these questions and help elucidate its mechanism.
In conclusion, CHDs were associated with a higher incidence of AMD, and the use of silymarin was associated with a reduced risk of AMD in patients with CHDs. However, a higher dose of silymarin was not associated with a signi cantly lower risk of AMD.

Declarations
Author contributions: Mu-Ming Chien and Hsiu-Chen Lin contributed to conception and design, analysis and interpretation of data, drafting the article and revising it critically for important intellectual content, and nal approval of the version to be published. Li-Fen Lee contributed to conception and design, analysis and interpretation of data, and nal approval of the version to be published. Joseph Jordan Keller partly contributed to acquisition of data, analysis and interpretation of data, revising it critically for important intellectual content and nal approval of the version to be published. Li-Hsuan Wang contributed to conception and design, analysis and interpretation of data, drafting the article, revising it critically for important intellectual content and nal approval of the version to be published.