Metabolomic profiling of serum and tongue coating of pregnant women with intrahepatic cholestasis in pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is associated with an increased risk of cesarean section and adverse fetal outcomes. Currently, ICP diagnosis depends largely on serum levels of bile acids and lacks sensitivity and specificity for accurate diagnosis. Tongue diagnosis is an important diagnostic tool in traditional Chinese medicine (TCM) and is used in our clinic as complementary treatment and personalized medicine for ICP. However, the molecular basis of the manifestation of greasy white tongue coatings in ICP remains unknown. In this study, we performed untargeted metabolomic profiling of the serum, tongue coating, and saliva of 66 pregnant women, including 22 with ICP. The metabolomic profiles of the serum and tongue coatings showed marked differences between the two clinical groups. Forty-six differentially abundant metabolites were identified, and their relative concentrations correlated with total bile acid levels. These differential metabolites included bile acids, lipids, microbiota- and diet-related metabolites, and exposomes. Conventional biochemical markers, including serum aminotransferases and bilirubin, were not significantly increased in the ICP group, whereas the total cholesterol and triglyceride levels were significantly increased as early as the first trimester. Our data provide insights into the pathophysiology of ICP and implicate the gut-liver axis and environmental exposure. Tongue coating has the potential to be a non-invasive diagnostic approach. Further studies are required to validate the clinical utility of these findings.

Mitigation of intrahepatic cholestasis induced by 17α-ethinylestradiol via nanoformulation of Silybum marianum L.

BACKGROUND: Cholestasis is an important predisposing factor for hepatocyte damage, liver fibrosis, primary biliary cirrhosis, and even liver failure. Silybum marianum L. (SM) plant is used in teas or eaten in some countries due to its antioxidant and hepatoprotective properties. Because of its low and poor oral bioavailability, so we improve the therapeutic activity of Silybum marianum L. extract (SM) by studying the potential effects of nanoformulation of Silybum marianium L. extract (nano-SM) on 17α-ethinylestradiol (EE)-induced intrahepatic cholestasis. METHODS: Thirty female Sprague-Dawley rats were divided into 5 groups (6 rats/group). Group I: Rats were received the treatment vehicle and served as normal group. Group II:Rats were injected daily with EE (10 mg/kg) for five successive days. Group III-V: Rats were injected daily with EE (10 mg/kg) and treated with either Ursodeoxycholic acid (UDCA) (40 mg/kg), SM (100 mg/kg) and nano-SM (100 mg/kg) orally once/day throughout the trialfor five successive days, respectively. RESULTS: Nano-SM greatly dampened the increase in serum levels of total and direct bilirubin, alanine aminotransaminase, aspartate aminotransaminase, and alkaline phosphatase caused by EE. Furthermore, nano-SM increased the hepatic contents of reduced glutathione (GSH) and catalase (CAT) and also upregulated the relative hepatic gene expressions of Rho-kinase (ROCK-1), myosin light chain kinase (MLCK), and myosin phosphatase target subunit (MYPT1) compared to the EE-induced group. Administration of nano-SM reduced hepatic lipid peroxidation and downregulated the relative hepatic expressions of the nuclear factor-kappa B (NF-Ò¡B) and interleukin-1ß (IL-1ß). In addition, nano-SM improved the histopathological changes induced by EE. CONCLUSION: Nano-SM possessed a superior effect over SM, which can be considered an effective protective modality against EE-induced cholestatic liver injury through its antioxidant, anti-inflammatory activities, and enhancing bile acid (BA) efflux.

Pathogenesis and Management of Citrin Deficiency.

Citrin deficiency (CD) is a hereditary disorder caused by SLC25A13 mutations that manifests as neonatal intrahepatic cholestasis caused by CD (NICCD), failure to thrive and dyslipidemia caused by CD (FTTDCD), and adult-onset type 2 citrullinemia (CTLN2). Citrin, an aspartate-glutamate carrier primarily expressed in the liver, is a component of the malate-aspartate shuttle, which is essential for glycolysis. Citrin-deficient hepatocytes have primary defects in glycolysis and de novo lipogenesis and exhibit secondarily downregulated PPARα, leading to impaired ß-oxidation. They are unable to utilize glucose and free fatty acids as energy sources, resulting in energy deficiencies. Medium-chain triglyceride (MCT) supplements are effective for treating CD by providing energy to hepatocytes, increasing lipogenesis, and activating the malate-citrate shuttle. However, patients with CD often exhibit growth impairment and irreversible brain and/or liver damage. To improve the quality of life and prevent irreversible damage, MCT supplementation with a diet containing minimal carbohydrates is recommended promptly after the diagnosis.

The IDI1/SREBP2 axis drives intrahepatic cholestasis and is a treatment target of San-Huang-Cai-Zhu formula identified by sequencing and experiments.

San-Huang-Chai-Zhu formula (SHCZF), originates from Da-Huang-Xiao-Shi decoction (DHXSD) for the treatment of jaundice as recorded in the Chinese traditional Chinese medicine book Jin Gui Yao Lue. In the clinic, SHCZF has been used to treat cholestasis-related liver disease by improving intrahepatic cholestasis, but the treatment mechanism has not been elucidated. In this study, 24 Sprague-Dawley (SD) rats were randomly assigned to the normal, acute intrahepatic cholestasis (AIC), SHCZF, and ursodeoxycholic acid (UDCA) groups. In addition, 36 SD rats were divided into dynamic groups, namely, normal 24 h, AIC 24 h, normal 48 h, AIC 48 h, normal 72 h, and AIC 72 h groups. Alpha-naphthylisothiocyanate (ANIT) was used to induce an AIC rat model. Serum biochemical indices and hepatic pathology were detected. Part of the hepatic tissues was used for sequencing, and others were used for subsequent experiments. Sequencing data combined with bioinformatics analysis were used to screen target genes and identify the mechanisms of SHCZF in treating AIC rats. Quantitative real-time PCR (qRT-PCR) and Western blotting (WB) were used to detect the RNA/Protein expression levels of screened genes. Rats in the dynamic group were used to determine the sequence of cholestasis and liver injury. High-performance liquid chromatography (HPLC) was used to determine the representative bioingredients of SHCZF. Sequencing and bioinformatics analysis suggested that IDI1 and SREBP2 are hub target genes of SHCZF to ameliorate ANTI-induced intrahepatic cholestasis in rats. The treatment mechanism is associated with the regulation of lipoprotein receptor (LDLr) to reduce cholesterol intake and 3-Hydroxy-3-Methylglutaryl-CoA reductase (HMGCR), and 3-Hydroxy-3-Methylglutaryl-CoA synthase 1 (HMGCS1) to decrease cholesterol synthesis. Animal experiments showed that SHCZF significantly reduced the expression levels of the above genes and proinflammatory cytokine lipocalin 2 (LCN2), inflammatory cytokines interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), thereby improving intrahepatic cholestasis and inflammation and liver injury.

Consecutive baicalin treatment relieves its accumulation in rats with intrahepatic cholestasis by increasing MRP2 expression.

Baicalin, an important flavonoid isolated from Scutellaria baicalensis Georgi, is a Chinese herb widely used in clinical practice. We previously reported the in vivo accumulation of baicalin in rats with intrahepatic cholestasis (IHC) after a single dose. However, the effects of the long-term administration of baicalin on its pharmacokinetics are unknown. Thus, we investigated the disposition of baicalin in normal rats and those with IHC after single and multiple consecutive administrations. In addition, we further investigated the effect of baicalin on multidrug resistance protein 2 (MRP2) in vivo to explore the underlying mechanism. In our study, the liquid chromatography-mass spectrometry (LC-MS) method established to determine baicalin concentrations in rat blood was simple, specific, and with linearity (R2 = 0.9980) in the range of 1.01-506.00 µg/mL. The relative standard deviations (RSD) for intra-day and inter-day precision were not more than 10.55%, and the intra-day and inter-day accuracies were 94.94%-109.13%. The recovery rate and stability were in line with the requirements of the quantitative analysis of biological samples as stated in the Chinese Pharmacopoeia (2020 Edition). Compared with that in normal rats, the Cmax and t1/2 increased significantly in EE-induced rats with IHC, whereas the clearance (CL) decreased after a single administration of baicalin. However, the area under the curve decreased, CL increased, and the t1/2 was shortened after the continuous administration of baicalin in the IHC rat model compared with the single administration of baicalin, and the pharmacokinetic characteristics were similar to those in normal rats. Moreover, MRP2 expression increased in rats with IHC with the continuous administration of baicalin. Continuous baicalin intervention could effectively reduce its accumulation in rats with IHC, and the mechanism may be attributed to its enhancement of MRP2 expression.

[Study on protective effect of Chaihu Shugan Powder against liver injury in rats with intrahepatic cholestasis by regulating FXR/Nrf2/ARE pathway].

This study aims to investigate the effect of Chaihu Shugan Powder(CHSG) on liver injury in rats with intrahepatic cholestasis by regulating farnesoid X receptor(FXR)/nuclear factor erythroid-2-related factor(Nrf2)/antioxidant response element(ARE) pathway. Eighty-four SD rats were classified into normal group, model group, CHSG-L group(0.5 g·kg~(-1)), CHSG-H group(2.5 g·kg~(-1)), ursodeoxycholic acid group(UDCA group, 100 mg·kg~(-1)), CHSG-H+sh-NC group(2.5 g·kg~(-1) CHSG+subcutaneous injection of sh-NC lentivirus), CHSG-H+sh-FXR group(2.5 g·kg~(-1) CHSG+subcutaneous injection of sh-FXR lentivirus), with 12 rats in each group. Rats were treated with corresponding drugs except for the normal group and the model group, once a day, for 7 days. On 5 th day, rats, except the normal group, were given α-naphthalene isothiocyanate(ANIT) at a dose of 100 mg·kg~(-1), once a day for 3 days to induce intrahepatic cholestasis, and the normal group was given the same amount of normal saline. Rats were anesthetized 1 h after the last administration and the 2 h bile flow was measured. Aeroset chemistry analyzer was employed to detect the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), and total bile acid(TBA) in rat serum. Based on hematoxylin and eosin(HE) staining, the pathological changes of rat liver tissue were observed. Glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in rat liver tissue homogenate were monitored with corresponding kits. Western blot was used to detect the expression of FXR, Nrf2, and heme oxygenase-1(HO-1) proteins in rat liver tissue. Compared with the normal group, the model group showed many spots or concentrated necrotic areas in the liver tissue, infiltration of a large number of inflammatory cells, swelling liver cells with nuclear shrinkage. The 2 h bile flow, levels of GSH-Px and SOD, and relative expression of FXR, Nrf2, and HO-1 proteins were significantly lower, and the levels of ALT, AST, TBIL, TBA and MDA were significantly higher in the model group than in the normal group. Compared with the model group, CHSG-L group, CHSG-H group, and UDCA group demonstrated significant alleviation of pathological damage of the liver tissue, significantly high 2 h bile flow, levels of GSH-Px and SOD, and expression of FXR, Nrf2 and HO-1 proteins, and significantly low levels of ALT, AST, TBIL, TBA and MDA. Compared with the CHSG-H group, the CHSG-H+sh-FXR group had worse liver pathological damage, significantly low levels of 2 h bile flow, levels of GSH-Px and SOD, and expression of FXR, Nrf2, and HO-1 proteins, and significantly high levels of ALT, AST, TBIL, TBA, and MDA. CHSG may protect against liver injury in rats with intrahepatic cholestasis by activating the FXR/Nrf2/ARE pathway.

Oleanolic acid alleviates ANIT-induced cholestatic liver injury by activating Fxr and Nrf2 pathways to ameliorate disordered bile acids homeostasis.

BACKGROUND: Cholestasis is a clinical syndrome with high incidence and few effective treatments. Oleanolic acid (OA) is a triterpenoid compound with anti-cholestatic effects. Studies using bile duct ligation or lithocholic acid modeling have shown that the alleviating effect of OA on cholerosis is related to the regulation of nuclear factor erythroid 2 related factor (Nrf2) or farnesoid X receptor (Fxr). PURPOSE: This study aims to investigate the underlying mechanism of OA against alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury based on Nrf2 and Fxr dual signaling pathways. METHODS: The ANIT-induced rats model was used with or without OA treatment. Serum biochemical indexes, liver histopathological changes and glutathione level were examined. Bile acids (BAs) targeted metabolomics based on UHPLC-MS/MS were performed. siRNA, RT-qPCR and western blot analysis were used to prove the role of Fxr and Nrf2 pathway in OA's anti-cholestatic liver injury in vivo and in vitro. RESULTS: OA significantly alleviated ANIT-induced liver injury in rats, reduced primary bile acids, accelerated metabolism of BAs and reduced the intrahepatic accumulation of BAs. The expressions of bile salt export pump (Bsep), Na+-taurocholic cotransport polypeptide (Ntcp), UDP-glucuronyl transferase 1a1 (Ugt1a1) and Fxr in rat liver were markedly up-regulated, the activation of Nrf2 was promoted, and the expression of cholesterol 7α-hydroxylase (Cyp7a1) was decreased after OA treatment. Moreover, Fxr or Nrf2 silencing attenuated the regulation of OA on BAs homeostasis related transporters and enzymes in rat primary hepatocytes. CONCLUSION: OA may regulate BAs-related transporters and metabolic enzymes by activating Fxr and Nrf2 pathways, thus alleviating the cholestatic liver injury induced by ANIT.

Comparative Evidence for Intrahepatic Cholestasis of Pregnancy Treatment With Traditional Chinese Medicine Therapy: A Network Meta-Analysis.

Background: Intrahepatic cholestasis of pregnancy (ICP) seriously threatens the health of pregnant women and newborns. A various number of Chinese prescriptions and patent medicines combined with ursodeoxycholic acid (UDCA) are used for treating ICP in China. However, there are still many doubts in choosing the suitable therapeutic drugs for the treatment of ICP in clinical practice. Methods: Several electronic databases, including PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure (CNKI), China Biology Medicine disc (CBM), Wanfang, and VIP, were comprehensively searched from the database inception to February 22, 2021. Randomized controlled trials (RCTs) reporting the use of UDCA only, Chinese prescriptions plus UDCA, and patent medicine plus UDCA for the treatment of ICP were collected according to their inclusion and exclusion criteria. Cochrane Reviewers' Handbook version 5.2 was applied for the risk assessment of the included trials. STATA 16.0 software was used for network meta-analysis (NMA). The pruritus score and the serum levels of total bile acid (TBA), alanine aminotransferase (ALT), and aspartate transaminase (AST) in ICP patients served as the primary outcomes. Moreover, this study had been registered in PROSPERO (https://www.crd.york.ac.uk/PROSPERO/#joinuppage), and the registration number is CRD42020188831. Results: Thirty-eight RCTs comprising 3,841 patients meeting the inclusion criteria were included in the network meta-analysis. The NMA results showed that compared with UDCA used alone, Yinchenhao decoction (seven different Chinese prescriptions or patent medicines) plus UDCA dramatically alleviated the primary outcomes of ICP, including the pruritus score, as well as the serum levels of TBA, ALT, and AST. The NMA results showed that the optimal drug ratio for the treatment of ICP was different from the dosage ratio of traditional Yinchenhao decoction. Significantly, the intervention plan f (IP-f) group [the similar prescription of Yinchenhao decoction 2 (Artemisia capillaris Thunb >15 g, Gardenia >9 g, and Rhubarb <5 g) + UDCA] was the best therapeutics among the eight therapies. Conclusion: Overall, the combined use of Chinese prescriptions or patent medicine with UDCA was generally better than UDCA used alone. The dose of IP-f might be a beneficial therapeutic method for the clinical medication of ICP. Clinical Trail Registration: https://www.crd.york.ac.uk/, identifier CRD42020188831.

Metabolic basis and treatment of citrin deficiency.

Citrin deficiency is a hereditary disorder caused by SLC25A13 mutations and manifests as neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD), and adult-onset type II citrullinemia (CTLN2). Citrin is a component of the malate-aspartate nicotinamide adenine dinucleotide hydrogen (NADH) shuttle, an essential shuttle for hepatic glycolysis. Hepatic glycolysis and the coupled lipogenesis are impaired in citrin deficiency. Hepatic lipogenesis plays a significant role in fat supply during growth spurt periods: the fetal period, infancy, and puberty. Growth impairment in these periods is characteristic of citrin deficiency. Hepatocytes with citrin deficiency cannot use glucose and fatty acids as energy sources due to defects in the NADH shuttle and downregulation of peroxisome proliferator-activated receptor α (PPARα), respectively. An energy deficit in hepatocytes is considered a fundamental pathogenesis of citrin deficiency. Medium-chain triglyceride (MCT) supplementation with a lactose-restricted formula and MCT supplementation under a low-carbohydrate diet are recommended for NICCD and CTLN2, respectively. MCT supplementation therapy can provide energy to hepatocytes, promote lipogenesis, correct the cytosolic NAD+ /NADH ratio via the malate-citrate shuttle and improve ammonia detoxification, and it is a reasonable therapy for citrin deficiency. It is very important to administer MCT at a dose equivalent to the liver's energy requirements in divided doses with meals. MCT supplementation therapy is certainly promising for promoting growth spurts during infancy and adolescence and for preventing CTLN2 onset. Intravenous administration of solutions containing fructose is contraindicated, and persistent hyperglycemia should be avoided due to glucose intoxication for patients receiving hyperalimentation or with complicating diabetes.

Oral herbal medicine for women with intrahepatic cholestasis in pregnancy: a systematic review of randomized controlled trials.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy complication whose range has been calculated to be between 0.01 and 15.6% all around the world. We wanted to systematically evaluate the effect and safety of oral herbal medicine on treatment for ICP. METHODS: Details of the methods could be found in the registered protocol on PROSPERO (CRD42018096013). Trials assessing the effectiveness of herbal medicine for ICP were searched from seven electronic databases from inception to 28th February 2020. RevMan 5.3 software was used to perform all statistical analysis. Meta-analysis, additional analysis, Trial Sequential Analysis (TSA) and Grading of Recommendations Assessment, Development and Evaluation (GRADE) were conducted if data permitted. RESULTS: Totally 43 randomized controlled trials with 3556 patients were included. Meta-analysis showed potential good adjunctive effect of herbal medicine on decreasing the pruritus scores (MD -0.58, 95% CI - 0.79 to - 0.36), the serum TBA scores (MD - 3.99 µmol/L, 95% CI - 4.24 to - 3.74) on the basis with Ursodesoxycholic acid. Compared to the medicine alone, significantly lower incidence of fetal distress (RR 0.41, 95% CI 0.32 to 0.51), asphyxia neonatorum (RR 0.35, 95%CI 0.25 to 0.49), cesarean section (RR 0.73, 95% CI 0.63 to 0.85), postpartum hemorrhage (RR 0.45, 95% CI 0.28 to 0.72) were observed in the combination group. But the comparison between herbal medicine and medicine showed inconsistent results among trials. Insufficient information could be used to evaluate the safety of herbal medicine for ICP. CONCLUSION: This review found the current evidence may support the effectiveness of combination of herbal medicine and conventional medicine for decreasing the maternal pruritus scores, the serum TBA, and the number of fetal distress, or asphyxia neonatorum events related to this condition (which was supported by TSA results). Since there were obvious statistical and clinical heterogeneity among trials, and the methodological quality of the included studies was poor, the level of the evidence could only be defined as "very low" according to the GRADE criteria. Further high quality studies are still needed to testify the effectiveness and safety of herbal medicine for ICP.

The effect of emodin on liver disease -- comprehensive advances in molecular mechanisms.

Liver injury could be caused by a variety of causes, including alcohol, drug poisoning, autoimmune overreaction, etc. In the period of liver injury, hepatic stellate cells (HSCs) will be activated and produce excessive extracellular matrix (ECM). If injury cannot be suppressed, liver injury will develop into fibrosis, even cirrhosis and liver cancer. It is reported that some monomer components extracted from traditional Chinese medicine have better effects on protecting liver. Emodin, an anthraquinone compound extracted from the traditional Chinese medicine RHEI RADIX ET RHIZOMA, has anti-inflammatory, antioxidant, liver protection and anti-cancer effects, and can prevent liver injury induced by a variety of factors. By searching literatures related to the liver protection of emodin in PUBMED, SINOMED, EBM and CNKI databases, it was found that emodin could inhibit the production and promote the secretion of bile acids, and have a protective effect on intrahepatic cholestasis. Also, emodin reduce collagen synthesis and anti-hepatic fibrosis by inhibiting oxidative stress, TGF-ß/Smad pathway and HSCs proliferation, and promoting apoptosis of HSCs. Emodin can also regulate lipid metabolism and regulate the synthesis and oxidation of lipids and cholesterol to protect the nonalcoholic fatty liver. Besides, emodin can induce the apoptosis of hepatocellular carcinoma cells by acting on the death receptor pathway and mitochondrial apoptosis pathway, thus inhibiting the development of hepatocellular carcinoma. Moreover, emodin can modulate immunity and improve immune rejection in liver transplantation animals. In conclusion, emodin has a good effect on liver protection, but further experimental data are needed to verify it.

Coenzyme Q 10 supplementation: A potential therapeutic option for the treatment of intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy specific liver disease characterized by pruritus, elevated serum bile acids and abnormal liver function that may be associated with severe adverse pregnancy outcomes. We previously reported that plasma coenzyme Q10 (CoQ10) is decreased in women with ICP as it is its analogue coenzyme Q9 (CoQ9) in rats with ethinyl estradiol (EE)-induced cholestasis. The aim of the present study was to evaluate the possible therapeutic role of CoQ10 in experimental hepatocellular cholestasis and to compare it with ursodeoxycholic acid (UDCA) supplementation. Bile acids, CoQ9, CoQ10, transaminases, alkaline phosphatase, retinol, α-tocopherol, ascorbic acid, thiobarbituric acid reactive substances, carbonyls, glutathione, superoxide dismutase and catalase were assessed in plasma, liver and/or hepatic mitochondria in control and cholestatic rats supplemented with CoQ10 (250 mg/kg) administered alone or combined with UDCA (25 mg/kg). CoQ10 supplementation prevented bile flow decline (P < 0.05) and the increase in serum alkaline phosphatase and bile acids, particularly lithocholic acid (P < 0.05) in cholestatic rats. Furthermore, it also improved oxidative stress parameters in the liver, increased both CoQ10 and CoQ9 plasma levels and partially prevented the fall in α-tocopherol (P < 0.05). UDCA also prevented cholestasis, but it was less efficient than CoQ10 to improve the liver redox environment. Combined administration of CoQ10 and UDCA resulted in additive effects. In conclusion, present findings show that CoQ10 supplementation attenuated EE-induced cholestasis by promoting a favorable redox environment in the liver, and further suggest that it may represent an alternative therapeutic option for ICP.

Ginsenoside Rg1 alleviates ANIT-induced intrahepatic cholestasis in rats via activating farnesoid X receptor and regulating transporters and metabolic enzymes.

Ginsenoside Rg1 is an active ingredient extracted from the roots of ginsenoside, and an α-naphthylisothiocyanate (ANIT)-induced rat model of intrahepatic cholestasis was used to investigate the protective effect of Rg1 on cholestasis. 48 SD male rats were randomly divided into 6 groups: control group, model group, UDCA group (ursodeoxycholic acid), low-dose Rg1 group (10 mg/kg), medium-dose Rg1 group (20 mg/kg) and high-dose Rg1 group (40 mg/kg). The model group, the UDCA group and all the Rg1 group were then intragastrically administered with 80 mg/kg ANIT, and the control group were given equal volume of olive oil. Then the pathological changes in liver tissue were observed, the secretion of bile in the bile duct was measured, and the biochemical markers in serum were quantified, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glutamyl transfer peptidase (GTP) and the content of total bilirubin (TBIL), direct bilirubin (DBIL), total bile acid (TBA). The contents of inflammatory mediators in serum were quantified, including tumor necrosis factor (TNF-α), γ-interferon (IFN-γ) and interleukin-1ß (IL-1ß). The contents of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in liver homogenate were quantified. Expression of farnesoid X receptor (FXR), transporters and metabolic enzymes in liver tissue was monitored. Rg1 treatment improved liver tissue pathological damage, promoted bile secretion and significantly reduced serum levels of the intrahepatic cholestasis markers ALT, AST, ALP, GTP, TBIL, DBIL and TBA. Rg1 increased the activity of SOD and GSH-Px in liver homogenate, while, reducing the serum levels of MDA and inflammatory mediators. Rg1 also regulated the expression of FXR, bile acid transporters and metabolic enzymes. Overall, Rg1 alleviated liver injury by improving secretion of bile and normalizing the activity of enzymes in the serum. The protective mechanism appeared to be related to the activation of FXR and regulation of liver transporters and metabolic enzymes.

TianJiu therapy for α-naphthyl isothiocyanate-induced intrahepatic cholestasis in rats treated with fresh Ranunculus sceleratus L.

ETHNOPHARMACOLOGICAL RELEVANCE: TianJiu (TJ) therapy, one type of cold moxibustion, applies to specific acupuncture points with herbal patches of hot nature, providing a constant irritant to the skin until the presence of hyperemia and blistering. Traditional and clinical reports suggest that TJ is an effective therapy for the treatment of jaundice with fresh Ranunculus sceleratus L. (RS), in which protoanemonin is one of the main irritant constituents. However, the therapeutic effect of TJ treatment with fresh RS against intrahepatic cholestasis has not been studied in animal experiments. AIM OF THE STUDY: Present study was undertaken to investigate the effect of TJ treatment with fresh RS against intrahepatic cholestasis in rats and provide an experimental basis for the underlying mechanism of TJ therapy. MATERIALS AND METHODS: Male intrahepatic cholestatic Sprague-Dawley rats induced by 2% α-naphthylisothiocyanate (ANIT, 80 mg/kg B.W.) were treated by TJ therapy with fresh RS. The levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), direct bilirubin (DBIL), total bilirubin (TBIL), total bile acid (TBA), hepatic malondialdehyde (MDA) and nitric monoxide (NO), as well as hepatic body ratio, bile flow and hepatic histopathological assay were measured and evaluated to investigate the therapeutic effect of TJ treatment with fresh RS. Phytochemical analysis of fresh and dried RS was performed by gas chromatography-mass spectrometer (GC-MS). RESULTS: After TJ treatment with fresh RS, the abnormally elevated levels of serum AST, ALT, ALP, DBIL, TBIL and TBA, as well as hepatic MDA and NO at 108 h were reduced significantly (versus model group, P < 0.01). The hepatic body ratio, bile flow and hepatic pathological change of cholestatic rats at 108 h in TJ group were restored when compared with those of model group. Thirty-one compounds including lactones, flavonoids and phenolic acids were identified and determined by GC-MS analysis. The content of protoanemonin in fresh RS (9.49%) was about 25-fold higher than that in dried RS (0.38%). CONCLUSIONS: TJ treatment with fresh RS exhibited good therapeutic effect on ANIT-induced intrahepatic cholestasis in rats, which may be due to the attenuated oxidative stress in the liver tissue. It is rational for the ancients to choose fresh RS as TJ herbal patches because of its abundant protoanemonin with the character of irritant. The qualitative and quantitative results of GC-MS analysis provided the chemical basis of TJ therapy with fresh RS, which can be regarded as a simple and efficient method for the treatment of cholestasis hepatitis.

Quantitative Proteomics Reveals the Protective Effects of Huangqi Decoction Against Acute Cholestatic Liver Injury by Inhibiting the NF-κB/IL-6/STAT3 Signaling Pathway.

Intrahepatic cholestasis (IC) is a common syndrome that affects the liver, with treatment options being limited. Huangqi decoction (HQD), a classic herbal medicine, has shown protective effects against IC. In this study, isobaric tags for relative and absolute quantification-based quantitative proteomics was performed to investigate the potential mechanism of action of HQD on α-naphthylisothiocyanate (ANIT)-induced IC, resulting in 2796 quantified proteins across all samples, including 270 differentially expressed proteins under HQD treatment. Fuzzy c-means clustering analysis of these 270 proteins assigned the proinflammatory proteins, such as LCN2, SAA1, FGG, FGA, and FGB, to Cluster 1 (upregulated by ANIT, and downregulated by HQD). Functional bioinformatics and protein-protein interaction network analyses indicated that these proinflammatory proteins were involved in the STAT3 signaling pathway. Further real-time PCR and Western blot experiments confirmed that the expression of these proteins was consistent with the proteomic results. Moreover, HQD treatment decreased the phosphorylation of STAT3, induced by ANIT. Western blot experiments revealed that HQD treatment decreased phosphorylation of NF-κB and downregulated the expression of the inflammatory gene IL-6 and therefore inhibited the IL-6/STAT3 signaling pathway. In summary, the present study suggested that HQD may ameliorate acute cholestatic liver injury via inhibition of the NF-κB/IL-6/STAT3 signaling pathway.

Xiaoyan lidan formula ameliorates α-naphthylisothiocyanate-induced intrahepatic cholestatic liver injury in rats as revealed by non-targeted and targeted metabolomics.

Intrahepatic cholestasis is a clinical syndrome of liver damage with systemic circulation and intrahepatic accumulation of excessive toxic bile acids without effective therapeutic methods so far. Xiaoyan Lidan Formula (XYLDF), a traditional Chinese prescription, has long been clinically applied for hepatobiliary disorders due to cholestasis. But its mechanism remains unknown. In this study, a non-targeted metabolomics approach based on UHPLC-Q-TOF-MS/MS combined with a bile acids (BAs) - targeted metabolomics approach based on UHPLC-MS/MS were performed to elucidate the functional mechanisms of XYLDF on α-naphthylisothiocyanate(ANIT)-induced intrahepatic cholestasis rats. The results showed that a total of 39 endogenous metabolites with significant difference (VIP > 1.00, P < 0.05) were identified as biomarkers of ANIT-induced intrahepatic cholestasis in rats. After treatment by XYLDF, 22 biomarkers were reversed to the control-like levels, which involved in primary BA biosynthesis, bile acid metabolism and excretion, steroids metabolism, retinol metabolism, starch and sucrose metabolism, inter conversions between pentose and glucoronate as well as arachidonic acid metabolism. Meanwhile, the results of contents variation of BAs in liver and serum showed that both hydrophilic and hydrophobic BAs were markedly increased in the model rats, while XYLDF treatment could restore the increase induced by ANIT, which suggested that one of the mechanisms of XYLDF on cholestasis referred to regulation of metabolic homeostasis of cholic acid.

[Protective effects and action mechanism of extract from Tibetan medicine Yajima(Chrysosplenium nudicaule) on mice with intrahepatic cholestasis induced by ANIT].

Chrysosplenium nudicaule,Tibetan name " Yajima",is recorded as an effective medicine for the treatment of liver and gallbladder diseases by Tibetan Pharmacopoeia published in the past dynasties,but its traditional efficacy has not yet been investigated by means of modern pharmacological research methods. In this paper,the protective effect of extract of C. nudicaule(ECN) on liver injury in mice was observed by using the mice model of intrahepatic cholestasis(IC) induced by α-naphthyl isothiocyanate(ANIT) and the possible mechanism by which ECN work as the therapeutic agent was discussed. The results showed that the serum levels of AST,ALT,ALP,DBIL,TBIL and TBA of the model mice were notably reduced in dose-dependent manner(P<0. 01,P<0. 05). The activity of SOD and GSH-Px in the liver homogenate of mice was increased,while the content of MDA was decreased(P<0. 01,P<0. 05).Pathological examination of liver in mice showed that ECN could improve the pathological changes of liver tissue in mice. The mRNA expression level of genes related to bile acid metabolism were detected by RT-PCR and the results suggested that ECN could significantly increase the expression of genes such as BSEP,FXR and MRP2(P<0. 01,P<0. 05),meanwhile significantly reduce the expression of CYP7 A1(P<0. 01,P<0. 05). These results confirmed the protective effect of ECN on intrahepatic cholestasis-induced liver injury in mice,and indicated that the mechanism may be related to activating FXR and its target genes,reducing bile acid synthesis and increasing bile acid excretion. This study provides a modern pharmacological basis for the clinical application of Yajima in Tibetan medicine.

Protective effects and action mechanism of extract from Tibetan medicine Yajima(Chrysosplenium nudicaule) on mice with intrahepatic cholestasis induced by ANIT / 中国中药杂志

Chrysosplenium nudicaule,Tibetan name " Yajima",is recorded as an effective medicine for the treatment of liver and gallbladder diseases by Tibetan Pharmacopoeia published in the past dynasties,but its traditional efficacy has not yet been investigated by means of modern pharmacological research methods. In this paper,the protective effect of extract of C. nudicaule(ECN) on liver injury in mice was observed by using the mice model of intrahepatic cholestasis(IC) induced by α-naphthyl isothiocyanate(ANIT) and the possible mechanism by which ECN work as the therapeutic agent was discussed. The results showed that the serum levels of AST,ALT,ALP,DBIL,TBIL and TBA of the model mice were notably reduced in dose-dependent manner(P<0. 01,P<0. 05). The activity of SOD and GSH-Px in the liver homogenate of mice was increased,while the content of MDA was decreased(P<0. 01,P<0. 05).Pathological examination of liver in mice showed that ECN could improve the pathological changes of liver tissue in mice. The mRNA expression level of genes related to bile acid metabolism were detected by RT-PCR and the results suggested that ECN could significantly increase the expression of genes such as BSEP,FXR and MRP2(P<0. 01,P<0. 05),meanwhile significantly reduce the expression of CYP7 A1(P<0. 01,P<0. 05). These results confirmed the protective effect of ECN on intrahepatic cholestasis-induced liver injury in mice,and indicated that the mechanism may be related to activating FXR and its target genes,reducing bile acid synthesis and increasing bile acid excretion. This study provides a modern pharmacological basis for the clinical application of Yajima in Tibetan medicine.

Pharmacokinetic Characteristics of Baicalin in Rats with 17α-ethynyl-estradiol-induced Intrahepatic Cholestasis.

Baicalin is one of the main active ingredients of choleretic traditional Chinese medicine drug Radix Scutellariae. The aim of this study was to explore the pharmacokinetic characteristics of baicalin in rats with 17α-ethynylestradiol (EE)-induced intrahepatic cholestasis (IC) based on its choleretic effects. Firstly, rats were subcutaneously injected with EE solution (5 mg/kg, 0.25 mL/100 g) for 5 consecutive days to construct an IC model. Then the bile excretion rate, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bile acid (TBA) and pathological changes of the liver were detected. Secondly, after successfully modeling, the rats were intragastrically given baicalin solution (200 mg/kg) (n=6). Blood samples were collected from the tail vein at different time points after intragastric administration. The protective effects of low- (50 mg/kg), medium- (100 mg/kg) and high-dose (200 mg/kg) baicalin on the liver in IC rats were evaluated. The content of baicalin in plasma was detected by liquid chromatography-mass spectrometry/mass spectrometry and pharmacokinetics parameters were calculated. Pharmacodynamic results showed that low-, medium- and high-dose baicalin all significantly increased the average excretion rate of bile (P<0.05), and significantly decreased serum levels of ALT, AST and ALP and TBA (P<0.05). Meanwhile, HE staining showed that baicalin significantly relieved EE-induced hepatocyte edema and necrosis. Pharmacokinetic results exhibited that the absorption of baicalin in both IC and normal control rats showed bimodal phenomenon. Cmax, AU(0-t) and AUC(0-∞) of baicalin in IC rats were significantly higher than those of the normal control group (P<0.01). T1/2 of plasma baicalin in the model group was significantly extended to (11.09±1.84) h, with clearance dropping to 61.78% of that of the normal control group (P<0.01). The above results suggested that baicalin had protective effects on the liver of IC rats, accompanied by significantly increased in vivo exposure, delayed in vivo clearance and markedly alterative pharmacokinetic characteristics. This study provides a theoretical basis for further development of baicalin as a feasible drug for treating IC.

Effect of YHHJ on the expression of the hepatocellular bile acid transporters multidrug resistance-associated protein 2 and bile salt export pump in ethinylestradiol-induced cholestasis.

The herbal medicine Yin Huang Mixture (YHHJ; patent no. 200910031240.7) is an aqueous extract composed from various herbs, including Artemisia capillaries Thunb, Hypericum japonicum Thunb, Eucommia ulmoides Oliver, Rheum officinale Baill, Gardenia jasminoides Ellis, Poria cocos Wolf and Dictamnus dasycarpus Turcz. Previous studies have indicated that YHHJ treatment has a beneficial effect on ameliorating itching and reducing serum bile acid levels in patients with intrahepatic cholestasis of pregnancy (ICP). However, the molecular mechanisms of action of YHHJ in ICP have not been fully elucidated. Therefore, the present study investigated an experimental hepatocellular cholestasis model to explore the regulatory role of YHHJ on the expression of the bile acid carriers, multidrug resistance-associated protein 2 (MRP2) and the bile salt export pump (BSEP). Initially, 5 mg/kg/day 17-α ethinylestradiol (EE) was used to induce cholestasis in rats and primary isolated rat hepatocytes. Subsequently, 9 or 36 g/kg/day YHHJ water extract was administrated. Blood samples were collected and serum biochemical parameters of total bile acids (TBA), total bilirubin (TBil), alanine transaminase and aspartate aminotransferase levels were determined. Rat livers and primary isolated rat hepatocytes were obtained and the protein and mRNA expression levels of MRP2 and BSEP were analyzed by western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Results revealed that EE-induced hepatocellular cholestasis was associated with a significant increase in serum TBA and TBil levels, whereas, YHHJ treatment significantly reversed this effect (P<0.01). Further experiments on the molecular mechanism revealed that EE significantly decreased the expression of MRP2 and BSEP compared with the control group, whereas YHHJ treatment significantly upregulated MRP2 and BSEP expression in vivo and in vitro compared with no YHHJ treatment (P<0.01). In addition, to establish whether upregulation of MRP2 and BSEP protein expression levels resulted from increased expression of their respective mRNA, the mRNA expression levels were determined. Results indicated that YHHJ treatment significantly increased MRP2 and BSEP mRNA expression levels in EE-induced hepatocellular cholestasis compared with no YHHJ treatment (P<0.01). In conclusion, the present findings suggest that YHHJ effects EE-induced cholestasis and this process may be mediated through regulating hepatobiliary transporters, MRP2 and BSEP.