Bile acids in treatment of ocular disease
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Bear bile has been included in Asian pharmacopeias for thousands of years in treatment of several diseases, ranging from sore throat to hemorrhoids. The hydrophilic bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) are the major bile acids of bear bile. Both of these are available as synthetic formulations and are approved by the health administrations of several countries for treatment of cirrhosis and gallstones. This review briefly covers the use of bear bile in Traditional Chinese Medicine, bile acid physiology, approved use of UDCA and TUDCA in Western medicine, and recent research exploring their neuroprotective properties, including in models of ocular disease.
KeywordsNeuroprotection Apoptosis Retinal degeneration Bile acid Traditional Chinese medicine Tauroursodeoxycholic acid TUDCA Ursodeoxycholic acid UDCA
Bear bile use in traditional Chinese medicine
Bear bile, bile extract, and gallbladder have been used in Traditional Chinese Medicine (TCM) and other Asian pharmacopeia for thousands of years [1, 2, 3]. Bear bile is classified as a “cold” medicine used to treat diseases of “heat” that involve fevers and inflammation to detoxify the liver, to prevent convulsions and epileptic seizures, to dissolve kidney stones and gallstones, to shrink tumors and hemorrhoids, and to improve vision [1, 2, 3]. Its use continues in TCM and has spread with the establishment of Asian communities throughout the world . China’s State Pharmacopoeia lists 28 types of medicines containing bear bile, 15 of which are used in ophthalmology .
The Asian black bear (Ursus thibetanus or Selenarctos thibetanus), also known as the Moon bear due to its coat markings, is the traditional source species, but as Moon bear populations decline, other species such as the North American black bear are used, often with negative consequences on those populations . To address the issue of declining populations and in recognizing that trafficking in animal parts is a component of their rural economy (dried bear bile has a higher street value than cocaine ), controlled bear farming was established in China [2, 3, 4]. However, the harvesting of bear parts is considered by many to be cruel [4, 7], and it is illegal or greatly restricted in many countries [3, 4, 5, 6] (particularly the signatory nations of the Convention on International Trade in Endangered Species of Wild Fauna and Flora  and additionally in the USA by the Endangered Species Act ), and it is not clear that the farming is sustainable economically or politically [4, 7, 10]. Thus, there is great interest in replacing bear parts with herbal and synthetic substitutes in TCM [3, 4].
Bile acid physiology
Bile acids are biosynthesized from cholesterol in liver hepatocytes. They are largely contained in the enterohepatic circulation and are stored in the gallbladder. They are predominantly conjugated with glycine or taurine, allowing them to be fully ionized in the pH range of the small intestine . Bile acids have five major functions: elimination of cholesterol, micellination of dietary lipids to aid in their transport, stimulation of bile flow and biliary phospholipid secretion, and finally, negative feedback regulation of bile acid and cholesterol synthesis .
In humans, the principle primary bile acids are cholic acid and chenodeoxycholic acid (CDCA) and the principle secondary bile acids are deoxycholic acid (DCA) and lithocholic acid. Conversely, the biliary bile and the serum of some bears contain high proportions of ursodeoxycholic acid (UDCA), mainly occurring as its taurine conjugate tauroursodeoxycholic acid (TUDCA) [12, 13, 14, 15]. UDCA and conjugates comprise about 47% of the biliary bile make up in American black bear and up to 76% in Asiatic black bear . UDCA and TUDCA are found only in trace amounts (<1–5% of total biliary bile acid composition) in other vertebrates . Similar to biliary bile content, the taurine-conjugated form of UDCA comprises a large proportion (about 28%) of the circulating plasma levels of bile acids in American black bears . In comparison, TUDCA constitutes about 0.13% of the bile acid pool in human serum . As UDCA and TUDCA are the major constituents of bile from Asian black bears, these two hydrophilic bile acids have long been considered by many to be the bioactive components of bear bile [3, 4, 7, 10, 14].
Synthetic UDCA and TUDCA as therapeutics
UDCA and TUDCA were first synthesized from cholic acid in 1954 in Japan . Today UDCA is produced by several manufacturers worldwide and marketed under various trade names, including Ursodiol, Actigall, Ursosan, Urso, Urso Forte, and Deursil. UDCA is approved by several governments for treatment of liver diseases and for the dissolution of gallstones. In Italy, TUDCA is marketed as Taurolite and is used similarly. Typical oral dosing of UDCA is 7–15 mg/kg body weight daily. Both TUDCA and UDCA are well tolerated; the most common side effect is diarrhea in less than 2% of several populations tested [18, 19, 20].
Oral administration of UDCA results in UDCA and its conjugates becoming the proportionately dominant bile acids in biliary bile, and serum and plasma absolute concentrations increase over tenfold [21, 22, 23, 24, 25, 26]. About half of the dose is absorbed from the portal blood into liver via first pass extraction, where it is conjugated with glycine forming glycoursodeoxycholic acid (GUDCA) or taurine forming TUDCA [24, 27, 28, 29]. The percentage absorbed decreases with increasing dose such that absolute and proportional enrichment of the biliary bile with UDCA and conjugates plateaus at an as-yet undefined dose due to epimerization of UDCA to CDCA and endogenous bile acid synthesis [23, 27, 30, 31]. UDCA and conjugates are excreted from the biliary tree and resorbed through the enterohepatic circulation or metabolized to insoluble salts and excreted in the feces . Oral dosing with TUDCA produces similar changes in biliary bile acid composition and circulating bile acid composition and concentrations, but with higher proportions and concentrations of UDCA and conjugates, possibly due to reduced intestinal biotransformation of TUDCA, suggesting moderately enhanced bioavailability [18, 32].
The initial use of UDCA was for dissolution of gallstones; it dissolves gallstones by solubilizing cholesterol from the stone surface, and it reduces the saturation of cholesterol in the bile, enhancing cholesterol transport [24, 26, 33]. UDCA is still prescribed for gallstone dissolution in patients who cannot or do not want to undergo surgery [24, 27, 29].
The other major, institutionally approved use of UDCA or TUDCA is in treatment of cholestatic liver disease, especially primary biliary cirrhosis (PBC). The first report of the use of UDCA or TUDCA in treatment of liver diseases in Japanese patients came shortly after their laboratory synthesis was developed in the mid-1950s [24, 29, 34]. However, their use in treatment of liver disease increased greatly after it was fortuitously discovered that serum aminotransferase levels improved in patients with chronic active hepatitis who were receiving UDCA for gallstone dissolution [24, 35], a discovery that sparked a series of studies examining its efficacy in treatment of cholestatic liver disease [11, 24].
UDCA and TUDCA as antiapoptotic cytoprotectants
TUDCA and UDCA were originally thought to act largely through displacement of hepatotoxic, hydrophobic bile acids such as CDCA and DCA from the bile acid pool, thus preventing accumulation of toxic levels in enterohepatic circulation and so reducing the detergent properties of bile at the hepatocellular membrane [11, 24]. However, two groups nearly simultaneously discovered that UDCA may have more mechanistic actions than simply replacing toxic bile acids. Koga et al.  found that biliary epithelial cells (BECs) from PBC patient biopsy tissue had much less DNA fragmentation from patients treated with UDCA than from untreated patients, suggesting that BECs from treated patients undergo less apoptosis than those from untreated patients.
Rodrigues et al.  more directly tested for an antiapoptotic mechanism. They isolated mitochondria from rat liver and treated them with UDCA or DCA, a hydrophobic bile acid known to be cytotoxic. DCA increased mitochondrial swelling, calcein release, and reactive oxygen species (ROS) production, and decreased the mitochondrial membrane potential. UDCA treatment inhibited these effects. In the same study, they found that rats fed with high DCA diets had high concentrations of Bax protein in their mitochondria, an effect blocked by adding UDCA to their drinking water. This same group then demonstrated that UDCA treatment inhibited apoptosis induced by deoxycholic acid, okadaic acid, and transforming growth factor beta1 in rat hepatocytes in primary culture . They found that incubation with UDCA prevented cyctochrome c release, caspase activation, and cleavage of poly(ADP-ribose) polymerase. They also found that the apoptosis-inducing agents decreased mitochondrial transmembrane potential (MTP) and increased Bax translocation from the cytosol to the mitochondrial membrane, effects blocked by UDCA. From these combined studies, the authors concluded that UDCA treatment can directly inhibit changes in MTP and ROS production, thus preventing megapore opening, membrane potential transition, and subsequent initiation of an apoptotic cascade [37, 38].
In later experiments, the same group found that exposing isolated mitochondria to purified, recombinant Bax protein induced cyctochrome c release without permeability changes that result in mitochondrial swelling, that is, independently of the permeability transition pore opening. This effect in isolated mitochondria was prevented by co- or pre-incubation with TUDCA. Pre-incubating mitochondria with TUDCA, then washing out the media and replacing with fresh, produced the same results, indicating that the effect of TUDCA is not due to direct interaction between TUDCA and Bax protein in the incubation media . Thus, both UDCA and TUDCA have antiapoptotic effects on isolated mitochondria.
More recently, this group and others [40, 41] have demonstrated that in addition to effects at the mitochondrial membrane, UDCA and TUDCA may have antiapoptotic effects by binding or otherwise activating nuclear steroid receptors. Following nuclear translocation, the hydrophilic bile acids appear to modulate the E2F-1/p53/Bax pathway as part of their antiapoptotic mechanism of action (reviewed in [42, 43]).
UDCA and TUDCA as neuroprotectants
Steer, Rodrigues, Kren, Low, and colleagues extended their studies in liver disease models to test the effects of UDCA or TUDCA treatment in models of neuronal disease and injury. Treatment of rat striatum in vivo or neuronal cells in culture with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, induces apoptosis and is considered a model of Huntington’s disease (HD). Co-incubation of an immortalized rat neuronal cell line or rat striatal cells in primary culture with TUDCA, UDCA, or GUDCA prevented 3-NP-induced apoptotic events such as MTP depolarization, mitochondrial-associated Bax translocation, and mitochondrial release of cytochrome c [44, 45]. Intraperotineal injection of rats with 3-NP caused loss of striatal volume, mitochondrial swelling, increased TUNEL signal in striatal tissue sections, and Rota-Rod sensorimotor task and cognitive testing deficits . All of these 3-NP-induced effects were largely or completely prevented for up to 6 months if TUDCA was co-administered with the neurotoxin . The authors tested whether TUDCA is also neuroprotective in a genetic model of HD. R6/2 mice that are transgenic for a causative CAG/polyglutamine repeat expansion of the human HD gene develop striatal intranuclear inclusions containing the proteins huntingtin and ubiquitin  followed by progressive neurological deficits similar to those of HD . IP injections of TUDCA starting at 6 weeks of age reduced striatal atrophy, decreased striatal apoptosis, resulted in fewer and smaller ubiquitinated neuronal intranuclear huntingtin inclusions, and improved locomotor and sensorimotor performance . Thus, systemic TUDCA treatment was found to be protective and antiapoptotic in cultured cells and in vivo in acute models and genetic models of HD.
Similarly, using in vivo cell culture and in vitro approaches, the same core group and colleagues found that treatment with UDCA or TUDCA slowed cell death in several neuronal disease models, including Alzheimer’s disease [48, 49, 50, 51, 52, 53, 54], Parkinson’s disease , acute hemorrhagic  and acute ischemic stroke , and neuronal glutamate toxicity .
Other laboratories have found that UDCA and TUDCA are protective in models of neuronal damage or degeneration. For example, incubation with UDCA prevents apoptosis in a model of cisplatin-induced sensory neuropathy, possibly by suppressing p53 accumulation . In an in vivo spinal cord injury model, rats injected systemically with TUDCA showed fewer apoptotic cord cells, less tissue injury, and better hind limb function than untreated control animals . Related, though not neuronal, UDCA and TUDCA are antiapoptotic cytoprotectants in ischemia/reperfusion and oxidative stress models of liver disease or injury [61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71] and models of heart disease [72, 73, 74, 75].
TUDCA as protectant in ocular models
Genetic models of photoreceptor degeneration
Other researchers have tested the effects of TUDCA in retinal degeneration models. TUDCA treatment slows retinal degeneration in s334ter-3 rats , a rat line that was genetically engineered to have a rhodopsin mutation identical to one common in autosomal dominant retinitis pigmentosa (ADRP) patients . These rats were injected daily from birth with TUDCA. Retinal sections from P5 and P10 rats showed that TUDCA treatment significantly decreased markers for reactive oxygen species, endoplasmic reticulum (ER) stress, and apoptosis. Retinal degeneration as assessed by morphology was also delayed in TUDCA-treated rats .
TUDCA treatment also slows retinal degeneration in P23H-3 rats [85, 86], another rat line that was genetically engineered to have an ADRP rhodopsin mutation . Rats were injected intraperitoneally (500 mg/kg body weight) once per week from P20 through 4 months old. In TUDCA- versus vehicle-treated rats, photoreceptor inner and outer segments, nuclei counts in the ONL, and the capillary retinal network were preserved. TUNEL signal was lower in TUDCA-treated rats compared to controls.
Induced models of photoreceptor degeneration
Ganglion cell death model
We also found that systemic TUDCA treatment slowed loss of retinal ganglion cells following optic nerve crush. Adult C57/Bl6 mice were treated as in the LIRD and rd10 mouse experiments for 10 days and anesthetized, and their optic nerves were transected [90, 91]. In some mice, a gelfoam sponge soaked in 3% Fluorogold was applied to the cut optic nerve stumps. Ten days after surgery, the number of fluorescing RGC somas seen in retina flatmounts declined by about 60% in vehicle-treated animals, a loss that was completely prevented in TUDCA-treated mice .
Thus, systemic treatment with TUDCA slows retinal degeneration in mouse and rat models, but it also has effects in other ocular tissues. Shinohara and colleagues report that TUDCA treatment prevents lens epithelial cell (LEC) death and cataract formation . Incubating cultured LEC with galactose, homocysteine, or tunicamycin induces ER stress and eventually death. However, pre- and co-incubation with TUDCA prevents this, even to a greater degree than treatment with the ER stress chaperones 4-phenylbutyric acid, trimethylamine N-oxide. Further, systemic injections of TUDCA significantly reduced lens epithelial cell death and partially delayed hypermature cataract formation in galactosemic rats, an effect accompanied by suppression of ER stress markers. Though others have reported that UDCA and TUDCA efficacy is mediated through alleviation of ER stress [93, 94, 95, 96], only Shinohara and colleagues have demonstrated that TUDCA acts as a chemical ER stress chaperone [83, 97].
The hydrophilic bile acids UDCA and TUDCA are cytoprotective in many disease models. Their exact mechanisms of action at the molecular level are not fully elucidated. Their cytoprotective actions are proposed to be mediated through actions at several points of the intrinsic and extrinsic apoptotic pathways and the ER stress response [42, 43, 92, 96, 98] from direct actions on mitochondrial and ER membranes through transcriptional regulation of both death and survival factors. Effects in ocular disease models have just begun to be tested and are clearly promising. However, almost no mechanistic data from ocular models have been reported. Indeed, some of the protective effects of TUDCA may be due to its taurine moiety suppressing formation of the apoptosome [99, 100]! Thus, in addition to continued preclinical testing for translational purposes, much work remains in determining how the hydrophilic bile acids have such marked protective effects in models of retinal degeneration, glaucoma, and cataract.
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