Learning from berberine: Treating chronic diseases through multiple targets

Although advances have been made, chemotherapy for chronic, multifactorial diseases such as cancers, Alzheimer’s disease, cardiovascular diseases and diabetes is far from satisfactory. Agents with different mechanisms of action are required. The botanic compound berberine (BBR) has been used as an over-the-counter antibacterial for diarrhea in China for many decades. Recent clinical studies have shown that BBR may be therapeutic in various types of chronic diseases. This review addresses BBR’s molecular mechanisms of action and clinical efficacy and safety in patients with type 2 diabetes, hyperlipidemia, heart diseases, cancers and inflammation. One of the advantages of BBR is its multiple-target effects in each of these diseases. The therapeutic efficacy of BBR may reflect a synergistic regulation of these targets, resulting in a comprehensive effect against these various chronic disorders. The safety of BBR may be due to its harmonious distribution into those targets. Although the single-target concept is still the principle for drug discovery and research, this review emphasizes the concept of a multiple target strategy, which may be an important approach toward the successful treatment of multifactorial chronic diseases.

Berberine (BBR; Figure 1) is a natural compound isolated from Chinese herbs such as Coptis chinensis and Berberis vulgaris [1]. BBR has a molecular weight (MW) of 336.37 Da and can be easily obtained from plants or through de novo synthesis [1,2]. It has anti-bacterial properties [3], and owing to its excellent safety profiles in humans, BBR has been utilized for many decades in China as an over-the-counter medicine for bacterial diarrhea. Recent studies have indicated that BBR may be effective in treating chronic, multifactorial diseases, including diabetes, hyperlipidemia, heart diseases, cancers and inflammatory diseases. In addition, laboratory studies have identified several molecules and signaling pathways that account for its therapeu-tic effects.
This review summarizes recent studies showing the clinical effects of BBR in diseases other than bacterially-caused diarrhea. Importantly, these studies indicate that the clinical effects of BBR are due to multiple molecules and/or mechanisms. Treatment of multifactorial chronic diseases with agents that regulate multiple molecular targets may be particularly effective in the future.

Diabetes
The glucose-lowering properties of BBR were first observed in 1986 [4]. Although the mechanism has not yet been fully determined, it was found to be related to several molecular mechanisms and signaling pathways. For example, BBR could activate the AMP-activated protein kinase (AMPK) pathway through mitochondrial inhibition [5], decreasing fasting blood glucose concentration. In addition, BBR could up-regulate the level of expression of the insulin receptor (InsR) gene in liver and muscle cells, in a protein kinase D (PKD)-dependent manner, restoring their insulin sensitivity [6]. BBR was shown to inhibit PTP1B and increase the phosphorylation of InsR, insulin receptor substrate 1 (IRS1) and Akt [7]. Moreover, BBR was found to inhibit liver gluconeogenesis while promoting the differentiation of adipocytes, resulting in a net increase in glucose utilization [8]. Recent studies have suggested that gut microbiota may be involved in the mechanism by which BBR reduces blood glucose and lipid concentrations [9,10].
Clinical reports have shown that BBR is safe and effective in treating type 2 diabetic patients [11]. In general, BBR alone lowered fasting blood glucose concentrations by 21%36%, comparable to the effects of metformin and rosiglitazone [11,13]. In addition, a clinical study in Italy suggested that BBR, when combined with standard glucose-lowering agents, such as metformin or sulfonylureas, demonstrated additive glucose-lowering efficacy in type 2 diabetic patients [12]. The main advantage of this botanic compound is its safety profile when compared with chemically synthesized glucose-lowering drugs such as rosiglitazone and metformin. For example, although the latter is not suitable for patients with chronic hepatitis and type 2 diabetes because it could lead to further deterioration in liver function, BBR was safe and effective, improving liver function in these patients while lowering blood glucose concentration [13]. In addition, animal experiments showed that BBR may have benefits in treating diabetic complications, such as endothelial dysfunction, diabetic nephropathy, and diabetic neuropathy [14].

Hyperlipidemia
BBR was first shown in 2004 to have lipid-lowering properties in animals as well as in hyperlipidemic patients [1]. Mechanistic studies have shown that BBR activates the extracellular-signal regulated kinase (ERK) pathway, stabilizing low-density-lipoprotein receptor (LDLR) mRNA and therefore increasing LDLR expression on the surface of hepatocytes [1]. This novel cholesterol-lowering mechanism differs from that of statin drugs. Detailed studies have shown that the activity of BBR is due to its effect on the responsive elements located in the LDLR mRNA 3′UTR region [1]. In addition, BBR was found to down-regulate the transcription of the gene encoding proprotein convertase subtilisin kexin 9 (PCSK9), a natural inhibitor of LDLR, suggesting another mechanism by which BBR increases LDLR expression [15]. BBR was also found to improve lipid dysregulation and prevent fatty liver by promoting the activity of AMPK [16], which has been reported to inhibit lipid synthesis [17].
Although to date no large-scale randomized double-blind clinical trials have been documented, the lipid-lowering effects of BBR have been validated by a number of independent clinical groups in and outside China. In general, BBR reduces total cholesterol concentrations by 13%31%, LDL cholesterol concentrations by 10%25%, and triglyceride concentrations by 20%35% [1,11,22], with one study reporting that BBR elevates HDL cholesterol concentrations [11]. The anti-lipid mechanism of BBR differs from that of statins, which increases LDLR expression by inhibiting HMG-CoA-reductase [18]. Thus, unlike statins, BBR has no adverse effects on liver or muscle tissue. This is of particular significance for Asian populations, which are at high risk for the adverse effects of statins [19]. BBR is also useful in controlling lipid concentrations in patients with liver diseases, such as hepatitis B and C and liver cirrhosis [20], as statins increase liver enzyme concentrations in some patients [21]. Interestingly, BBR had a more pronounced effect on triglyceride concentrations than did statins [22]. The clinical advantages of BBR may result from its effects on multiple pathways of lipid and glucose metabolism [23].

Heart diseases
Laboratory research has shown that BBR possesses positive inotropic, anti-arrhythmic and vasodilator properties related to the cardiovascular system [24] by, for example, prolonging the duration of ventricular action potential [24,25]. The effects of BBR are due, at least in part, to preferential blockage of the components of the delayed rectifying potassium current, I(Kr) and I(Ks) [25]. BBR may also act by stimulating the Na + -Ca 2+ exchanger [24]. Furthermore, BBR preferentially blocks the open state of hERG channels by interacting with specific residues [24]. Its vasodilator activity may result from undetermined multi-cellular mechanisms, which may be associated with the AMPK pathway and endothelial nitric oxide synthase [26]. Taken together, these cardiovascular effects of BBR support its clinical use in patients with heart failure or arrhythmias.
A randomized clinical trial tested the effects of BBR in 156 patients with chronic congestive heart failure [27]. Of these patients, 79 were and 77 were not treated with BBR, with all patients receiving conventional therapeutic regimens, consisting of angiotensin-converting enzyme inhibitors, digoxin, diuretics, and nitrates. The BBR-treated group showed significantly greater increases in left-ventricularejection-fraction (LVEF) and exercise capacity, significant improvements on the dyspnea-fatigue index, as well as decreased rates of ventricular premature complexes (VPCs) and long-term mortality (P<0.02). A second clinical trial yielded similar results [28].
Treatment of 100 arrhythmic patients with BBR (300 mg, qid) for 14 weeks resulted in a >89% reduction in premature beating in 62 patients, and >50% in the other 38 patients, indicating that BBR significantly reduced premature beating [29]. These results were later independently reproduced [30].

Cancer
BBR has been reported to have apoptotic activity in a variety of human tumor cell lines, including hepatoma, colon cancer, lung cancer, breast cancer, and leukemia cell lines [31]. BBR may act on cancer cell DNA, DNA replication enzymes (e.g., telomerase, DNA topoisomerase), matrix metalloproteinases (MMPs) in mitochondria, the Bcl2/Bax ratio, p53, and apoptosis inducing factor (AIF) [31]. Although, to our knowledge, the in vivo anticancer activities of BBR have not been documented to date, clinical studies have shown its potential in supporting radiation therapy for cancer. For example, a randomized clinical trial involving 78 cancer patients receiving abdominal or whole pelvic radiotherapy showed that, compared with patients who did not receive BBR, those pretreated or treated with BBR (300 mg kg 1 , tid) experienced significant decreases in the incidence and severity of radiation-induced acute intestinal symptoms (RIAIS; P<0.05) [32]. This suggests that BBR has a supporting effect in patients on radiation therapy. In another randomized double-blind trial [33], 90 patients with non-small cell lung cancer (NSCLC) were randomized 1 : 1 to radiation therapy plus BBR or radiation therapy plus placebo for six weeks. The rates of radiation-induced lung injury (RILI) six weeks and six months later were significantly reduced in the BBR group, whereas pulmonary function was significantly improved [33]. Despite these effects, the molecular mechanism of action of BBR remains unclear.
A clinical study assessed the effects in 21 breast cancer patients of cholesterol-lowering natural products on secondary dyslipidemia, which frequently persists after life style changes in patients on hormone-therapy following breast cancer (HT-BC) [34]. BBR plus Red Yeast Rice (AP), combined with diet, reduced total cholesterol, LDL and triglyceride concentrations and was well tolerated by these patients.
As inflammation is a key factor in the pathophysiological process of metabolic disorders, cardiovascular diseases and cancers [41], the anti-inflammatory activity of BBR may result in beneficial effects against these disorders. The anti-inflammatory effects of BBR in treating diabetes have been assessed in relative depth. BBR was found to block the development of type 1 diabetes in NOD mice through its anti-inflammatory and immune regulatory properties [42]. In addition, BBR inhibited the expression of proinflammatory cytokines in adipose tissues of db/db diabetic and obese mice [39]. A clinical report analyzed the anti-inflammatory effects of BBR in Chinese patients with type 2 diabetes [43], finding that BBR treatment for three months significantly reduced serum concentrations of pro-inflammatory cytokines, such as TNF-α and IL-6, as well as reducing blood glucose concentrations and restoring insulin sensitivity [43]. The anti-inflammatory effects of BBR have also been observed in patients with coronary heart disease. BBR treatment for 30 d, following standard intervention, reduced serum inflammatory markers, as well as lipid concentrations, in patients with acute coronary syndrome (ACS) [44].
These anti-inflammatory properties of BBR account, at least in part, for its pharmacological efficacy against diseases such as diabetes and cardiovascular disorders. However, its molecular mechanisms, the cellular signaling pathways involved and the clinical significance of this antiinflammatory activity of BBR require further study.

Other conditions
BBR also has effects on the nervous system. In animal models, for example, BBR treatment was shown to improve brain function (memory and learning capacity) and to relieve depression [4547]. As these activities have not yet been verified in humans, they are not further discussed in this review.

Perspectives
Single-target therapy has been the mainstay of molecule-based drug discovery, with the goal of reducing undesired side-effects of administered agents. This strategy has achieved success in the treatment of viral infections, such as HIV-1, HBV and HCV, using reverse transcriptase, protease, and integrase inhibitors. Drugs highly selective for single targets have also shown clinical benefits in treating non-infectious chronic diseases; however, their therapeutic efficacy is often transient, eventually failing because patients develop drug-resistance and/or side-effects.
A recent example is the withdrawal (and limited use) of thiazolidinediones (TZDs), a class of selective agonists of peroxisome proliferator-activated receptor gamma (PPARγ) with glucose-lowering effects in patients with type 2 diabetes [48]. Side-effects of TZDs were observed in the cardiovascular, hepatic, and urinary systems [4951]. The potency and selectivity of the TZD drugs toward PPARγ correlate positively with their side-effects [51]. The phenomenon was also seen with the cyclooxygenase-2 (Cox2) inhibitors, rofecoxib and valdecoxib [5254].
In contrast, BBR has various therapeutic effects, with multiple molecular mechanisms of action. For example, BBR has several activities in patients with energy-related metabolic disorders, such as hyperlipidemia and type 2 diabetes, acting through several pathways. BBR up-regulates LDLR by activating ERK and inhibiting PCSK9, reduces lipid synthesis and increases glucose consumption by activating AMPK, up-regulates InsR by activating PKD, and decreases insulin resistance by inhibiting PTP1B and mTOR. These molecules may be components of an intact network that regulates cellular energy metabolism. The effectiveness of BBR on metabolic disorders in patients has been widely documented by independent clinical groups, both inside and outside China; this therapeutic efficacy represents the synergistic effectiveness of these molecules or pathways regulated by BBR.
Two hypotheses may explain the multiple-target nature of BBR. First, as BBR is a small-molecular-weight compound, it may be a ligand that docks into pockets of a number of proteins. Second, BBR can bind to nucleic acids, such as dsRNA. As miRNA molecules play a key role in controlling the expression of different genes, BBR in its planar form [57] might bind to grooves on the 3D structure of RNA [55,56].
BBR has shown good safety results in human applications [1,11]. Despite having significant clinical efficacy, the potency of its biological activity against each target is considered moderate. A balanced distribution of the chemical energy of BBR into those targets may account for its safety, making "Yin and Yang" balanced in the body.
Drugs with multiple-targets, such as aspirin, steroids and metformin, are often used to successfully manage illnesses. Therefore, although the specific-or selective-target approach still dominates, a multiple-targeted strategy might be a promising avenue in drug discovery, particularly for chronic diseases associated with multiple factors.