Fernando A. Crocenzi

Silymarin as a new hepatoprotective agent in experimental cholestasis: new possibilities for an ancient medication.

Silymarin is a purified extract from milk thistle (Silybum marianun (L.) Gaertn), composed of a mixture of four isomeric flavonolignans: silibinin (its main, active component), isosilibinin, silydianin and silychristin. This extract has been empirically used as a remedy for almost 2000 years, and remains being used as a medicine for many types of acute and chronic liver diseases. Despite its routinely clinical use as hepatoprotectant, the mechanisms underlying its beneficial effects remain largely unknown. This review addresses in detail a number of recent studies showing a novel feature of silymarin as a hepatoprotective drug, namely: its anticholestatic properties in experimental models of hepatocellular cholestasis with clinical correlate. For this purpose, this review will cover the following aspects: 1. The chemistry of silymarin, including chemical composition and properties. 2. The current clinical applications of silymarin as a hepatoprotective agent, including the mechanisms by which silymarin is thought to exert its hepatoprotective properties, when known. 3. The physiological events involved in bile formation, and the mechanisms of hepatocellular cholestasis, focusing on cellular targets and mechanisms of action of drugs used to reproduce experimentally cholestatic diseases of clinical interest, in particular estrogens and monohydroxylated bile salts, where anticholestatic properties of silymarin have been tested so far. 4. The recent findings describing the impact of silymarin on normal bile secretion and its novel, anticholestatic properties in experimental models of cholestasis, with particular emphasis on the cellular/molecular mechanisms involved, including modulation of bile salt synthesis, biotransformation/depuration of cholestatic compounds, changes in transporter expression/activity, and evocation of signaling pathways.

Ursodeoxycholic acid in cholestasis: linking action mechanisms to therapeutic applications

UDCA (ursodeoxycholic acid) is the therapeutic agent most widely used for the treatment of cholestatic hepatopathies. Its use has expanded to other kinds of hepatic diseases, and even to extrahepatic ones. Such versatility is the result of its multiple mechanisms of action. UDCA stabilizes plasma membranes against cytolysis by tensioactive bile acids accumulated in cholestasis. UDCA also halts apoptosis by preventing the formation of mitochondrial pores, membrane recruitment of death receptors and endoplasmic-reticulum stress. In addition, UDCA induces changes in the expression of metabolizing enzymes and transporters that reduce bile acid cytotoxicity and improve renal excretion. Its capability to positively modulate ductular bile flow helps to preserve the integrity of bile ducts. UDCA also prevents the endocytic internalization of canalicular transporters, a common feature in cholestasis. Finally, UDCA has immunomodulatory properties that limit the exacerbated immunological response occurring in autoimmune cholestatic diseases by counteracting the overexpression of MHC antigens and perhaps by limiting the production of cytokines by immunocompetent cells. Owing to this multi-functionality, it is difficult to envisage a substitute for UDCA that combines as many hepatoprotective effects with such efficacy. We predict a long-lasting use of UDCA as the therapeutic agent of choice in cholestasis.

Impaired localisation and transport function of canalicular Bsep in taurolithocholate induced cholestasis in the rat.

Background: Taurolithocholate induced cholestasis is a well established model of drug induced cholestasis with potential clinical relevance. This compound impairs bile salt secretion by an as yet unclear mechanism. Aims: To evaluate which step/s of the hepatocellular bile salt transport are impaired by taurolithocholate, focusing on changes in localisation of the canalicular bile salt transporter, Bsep, as a potential pathomechanism. Methods: The steps in bile salt hepatic transport were evaluated in rats in vivo by performing pharmacokinetic analysis of 14C taurocholate plasma disappearance. Bsep transport activity was determined by assessing secretion of 14C taurocholate and cholyl-lysylfluorescein in vivo and in isolated rat hepatocyte couplets (IRHC), respectively. Localisation of Bsep and F-actin were assessed both in vivo and in IRHC by specific fluorescent staining. Results: In vivo pharmacokinetic studies revealed that taurolithocholate (3 μmol/100 g body weight) diminished by 58% canalicular excretion and increased by 96% plasma reflux of 14C taurocholate. Analysis of confocal images showed that taurolithocholate induced internalisation of Bsep into a cytosolic vesicular compartment, without affecting F-actin cytoskeletal organisation. These effects were reproduced in IRHC exposed to taurolithocholate (2.5 μM). Preadministration of dibutyryl-cAMP, which counteracts taurolithocholate induced impairment in bile salt secretory function in IRHC, restored Bsep localisation in this model. Furthermore, when preadministered in vivo, dibutyryl-cAMP accelerated recovery of both bile flow and bile salt output, and improved by 106% the cumulative output of 14C taurocholate. Conclusions: Taurolithocholate impairs bile salt secretion at the canalicular level. Bsep internalisation may be a causal factor which can be prevented by dibutyryl-cAMP.

Phosphoinositide 3‐kinase/protein kinase B signaling pathway is involved in estradiol 17β‐d‐glucuronide–induced cholestasis: Complementarity with classical protein kinase c

Estradiol 17β‐D‐glucuronide (E217G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance–associated protein 2 (Mrp2). We assessed whether phosphoinositide 3‐kinase (PI3K) is involved in E217G‐induced cholestasis. E217G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E217G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2‐Morpholin‐4‐yl‐8‐phenylchromen‐4‐one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E217G was extensively prevented by WM; this effect was fully blocked by the microtubule‐disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13‐tetrahydro‐13‐methyl‐5‐oxo‐12H‐indolo[2,3‐a]pyrrolo[3,4‐c]carbazole‐12‐propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E217G‐induced cholestasis. In isolated perfused rat liver, an intraportal injection of E217G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [3H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule‐dependent manner. Conclusion: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E217G through sustained internalization of canalicular transporters endocytosed via cPKC. (HEPATOLOGY 2010)

Hepatocellular transport in acquired cholestasis : new insights into functional, regulatory and therapeutic aspects

The recent overwhelming advances in molecular and cell biology have added enormously to our understanding of the physiological processes involved in bile formation and, by extension, to our comprehension of the consequences of their alteration in cholestatic hepatopathies. The present review addresses in detail this new information by summarizing a number of recent experimental findings on the structural, functional and regulatory aspects of hepatocellular transporter function in acquired cholestasis. This comprises (i) a short overview of the physiological mechanisms of bile secretion, including the nature of the transporters involved and their role in bile formation; (ii) the changes induced by nuclear receptors and hepatocyte-enriched transcription factors in the constitutive expression of hepatocellular transporters in cholestasis, either explaining the primary biliary failure or resulting from a secondary adaptive response; (iii) the post-transcriptional changes in transporter function and localization in cholestasis, including a description of the subcellular structures putatively engaged in the endocytic internalization of canalicular transporters and the involvement of signalling cascades in this effect; and (iv) a discussion on how this new information has contributed to the understanding of the mechanism by which anticholestatic agents exert their beneficial effects, or the manner in which it has helped the design of new successful therapeutic approaches to cholestatic liver diseases.

Regulation of Synthesis and Trafficking of Canalicular Transporters and its Alteration in Acquired Hepatocellular Cholestasis. Experimental Therapeutic Strategies for its Prevention

Bile formation is an osmotic process driven by the vectorial transport of actively transferred biliary components across the basolateral (sinusoidal) and apical (canalicular) hepatocyte membranes, the latter being the rate-limiting step of the overall blood-to-bile transfer. The ATP-binding cassette (ABC) superfamily of membrane transporters comprises novel ATP-dependent carriers that mediate canalicular transfer of several endogenous and exogenous substrates, and therefore play a key role in bile formation. Gene expression, as well as the balance between vesicular targeting and internalization of these transporters to/from the canalicular membrane are highly regulated processes. This balance is affected in several models of hepatocellular cholestasis, and these alterations may either initiate or perpetuate the cholestatic manifestations. This review describes the regulation of the normal activity of hepatocellular ABC transporters, focusing on the involvement of transcription factors and signaling pathways in the regulation of carrier synthesis, dynamic localization and phosphorylation status. Its alteration in different experimental models of cholestasis, such as those induced by estrogens, lipopolysaccharide (endotoxin), monohydroxylated bile salts and oxidative stress, is also reviewed. Finally, several experimental therapeutic approaches based upon the administration of compounds known/thought to induce carrier synthesis (e.g., protein synthesis inducers), to counteract etiological factors responsible for the cholestatic disease (e.g., corticoids in lipopolysaccharide-induced cholestasis) or to stimulate exocytic insertion of canalicular transporters (e.g., cAMP, silymarin or tauroursodeoxycholate) are described with respect to their ability to prevent cholestatic alterations; the role of signaling molecules as putative downstream mediators of their effects are also discussed.

G‐protein‐coupled receptor 30/adenylyl cyclase/protein kinase A pathway is involved in estradiol 17ß‐d‐glucuronide‐induced cholestasis

Estradiol‐17ß‐d‐glucuronide (E17G) activates different signaling pathways (e.g., Ca2+‐dependent protein kinase C, phosphoinositide 3‐kinase/protein kinase B, mitogen‐activated protein kinases [MAPKs] p38 and extracellular signal‐related kinase 1/2, and estrogen receptor alpha) that lead to acute cholestasis in rat liver with retrieval of the canalicular transporters, bile salt export pump (Abcb11) and multidrug resistance‐associated protein 2 (Abcc2). E17G shares with nonconjugated estradiol the capacity to activate these pathways. G‐protein‐coupled receptor 30 (GPR30) is a receptor implicated in nongenomic effects of estradiol, and the aim of this study was to analyze the potential role of this receptor and its downstream effectors in E17G‐induced cholestasis. In vitro, GPR30 inhibition by G15 or its knockdown with small interfering RNA strongly prevented E17G‐induced impairment of canalicular transporter function and localization. E17G increased cyclic adenosine monophosphate (cAMP) levels, and this increase was blocked by G15, linking GPR30 to adenylyl cyclase (AC). Moreover, AC inhibition totally prevented E17G insult. E17G also increased protein kinase A (PKA) activity, which was blocked by G15 and AC inhibitors, connecting the links of the pathway, GPR30‐AC‐PKA. PKA inhibition prevented E17G‐induced cholestasis, whereas exchange protein activated directly by cyclic nucleotide/MAPK kinase, another cAMP downstream effector, was not implicated in cAMP cholestatic action. In the perfused rat liver model, inhibition of the GPR30‐AC‐PKA pathway totally prevented E17G‐induced alteration in Abcb11 and Abcc2 function and localization. Conclusion: Activation of GPR30‐AC‐PKA is a key factor in the alteration of canalicular transporter function and localization induced by E17G. Interaction of E17G with GPR30 may be the first event in the cascade of signaling activation. (Hepatology 2014;59:1016–1029)

Preventive effect of silymarin against taurolithocholate-induced cholestasis in the rat

Increased amounts of monohydroxylated bile salts (BS) have been found in neonatal cholestasis, parenteral nutrition-induced cholestasis and Bylers disease, among others. We analyzed whether the hepatoprotector silymarin (SIL), administered i.p. at the dose of 100mg/kg/day for 5 days, prevents the cholestatic effect induced by a single injection of the model monohydroxylated BS taurolithocholate (TLC, 30 micromol/kg, i.v.) in male Wistar rats. TLC, administered alone, reduced bile flow, total BS output, and biliary output of glutathione and HCO(3)(-) during the peak of cholestasis (-75, -67, -81, and -80%, respectively, P<0.05). SIL prevented partially these alterations, so that the drops of these parameters induced by TLC were of only -41, -25, -60, and -64%, respectively (P<0.05 vs. TLC alone); these differences between control and SIL-treated animals were maintained throughout the whole (120 min) experimental period. Pharmacokinetic studies showed that TLC decreased the intrinsic fractional constant rate for the canalicular transport of both sulfobromophthalein and the radioactive BS [14C]taurocholate by 60 and 68%, respectively (P<0.05), and these decreases were fully and partially prevented by SIL, respectively. SIL increased the hepatic capability to clear out exogenously administered TLC by improving its own biliary excretion (+104%, P<0.01), and by accelerating the formation of its non-cholestatic metabolite, tauromurideoxycholate (+70%, P<0.05). We conclude that SIL counteracts TLC-induced cholestasis by preventing the impairment in both the BS-dependent and -independent fractions of the bile flow. The possible mechanism/s involved in this beneficial effect will be discussed.

GALACTOSAMINE PREVENTS ETHINYLESTRADIOL-INDUCED CHOLESTASIS

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17β-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17β-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17β-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17β-glucuronide. The evidence thus supports the hypothesis that EE 17β-glucuronide is involved in the pathogenesis of EE cholestasis.

Effect of silymarin on biliary bile salt secretion in the rat.

The effect of the hepatoprotector silymarin on bile secretion, with particular regard to bile salt secretion, was studied in Wistar rats. Silymarin (25, 50, 100, and 150 mg/kg/day, i.p., for 5 days) induced a dose-dependent increase in bile flow and bile salt secretion, the maximal effect being reached at a dose of 100 mg/kg/day (+17 and +49%, for bile flow and bile salt output, respectively; P < 0.05). Assessment of bile salt composition in bile revealed that stimulation of the bile salt secretion was accounted for mainly by an increase in the biliary secretion of beta-muricholate and, to a lesser extent, of alpha-muricholate, chenodeoxycholate, ursodeoxycholate, and deoxycholate. The maximum secretory rate (T(m)) of bile salts, as assessed by infusing the non-hepatotoxic bile salt tauroursodeoxycholate i.v. at stepwise-increasing rates, was not influenced by silymarin. The flavonolignan also increased the endogenous bile salt pool size (+53%, P < 0.05) and biliary bile acid excretion after bile acid pool depletion (+54%, P < 0.05), a measure of de novo bile salt synthesis. These results suggest that silymarin increases the biliary excretion and the endogenous pool of bile salts by stimulating the synthesis, among others, of hepatoprotective bile salts, such as beta-muricholate and ursodeoxycholate.