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Gajendiran P, Vega LI, Itoh K, Sesaki H, Vakili MR, Lavasanifar A, Hong K, Mezey E, Ganapathy-Kanniappan S. Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin. J Cell Mol Med 2018; 22:2210-2219. [PMID: 29397578 PMCID: PMC5867155 DOI: 10.1111/jcmm.13501] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2017] [Accepted: 11/18/2017] [Indexed: 12/17/2022] Open
Abstract
Activation of hepatic stellate cells (HSCs) is an integral component of the wound‐healing process in liver injury/inflammation. However, uncontrolled activation of HSCs leads to constant secretion of collagen‐rich extracellular matrix (ECM) proteins, resulting in liver fibrosis. The enhanced ECM synthesis/secretion demands an uninterrupted supply of intracellular energy; however, there is a paucity of data on the bioenergetics, particularly the mitochondrial (mito) metabolism of fibrogenic HSCs. Here, using human and rat HSCs in vitro, we show that the mito‐respiration, mito‐membrane potential (Δψm) and cellular ‘bioenergetic signature’ distinguish fibrogenic HSCs from normal, less‐active HSCs. Ex vivo, HSCs from mouse and rat models of liver fibrosis further confirmed the altered ‘bioenergetic signature’ of fibrogenic HSCs. Importantly, the distinctive elevation in mito‐Δψm sensitized fibrogenic HSCs for selective inhibition by mitotropic doxorubicin while normal, less‐active HSCs and healthy human primary hepatocytes remained minimally affected if not, unaffected. Thus, the increased mito‐Δψm may provide an opportunity to selectively target fibrogenic HSCs in liver fibrosis.
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Affiliation(s)
- Priya Gajendiran
- Division of Interventional Radiology, Russell H. Morgan Department of Radiology & Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Leonel Iglesias Vega
- Division of Interventional Radiology, Russell H. Morgan Department of Radiology & Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Kie Itoh
- Department of Cell Biology, School of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Hiromi Sesaki
- Department of Cell Biology, School of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Mohammad Reza Vakili
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada
| | - Afsaneh Lavasanifar
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada
| | - Kelvin Hong
- Division of Interventional Radiology, Russell H. Morgan Department of Radiology & Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Esteban Mezey
- Division of Gastroenterology and Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Shanmugasundaram Ganapathy-Kanniappan
- Division of Interventional Radiology, Russell H. Morgan Department of Radiology & Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Karthikeyan S, Potter JJ, Geschwind JF, Sur S, Hamilton JP, Vogelstein B, Kinzler KW, Mezey E, Ganapathy-Kanniappan S. Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model. Biochem Biophys Res Commun 2015; 469:463-9. [PMID: 26525850 DOI: 10.1016/j.bbrc.2015.10.101] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2015] [Accepted: 10/20/2015] [Indexed: 12/21/2022]
Abstract
Liver fibrosis and cirrhosis result from uncontrolled secretion and accumulation of extracellular matrix (ECM) proteins by hepatic stellate cells (HSCs) that are activated by liver injury and inflammation. Despite the progress in understanding the biology liver fibrogenesis and the identification of potential targets for treating fibrosis, development of an effective therapy remains elusive. Since an uninterrupted supply of intracellular energy is critical for the activated-HSCs to maintain constant synthesis and secretion of ECM, we hypothesized that interfering with energy metabolism could affect ECM secretion. Here we report that a sublethal dose of the energy blocker, 3-bromopyruvate (3-BrPA) facilitates phenotypic alteration of activated LX-2 (a human hepatic stellate cell line), into a less-active form. This treatment-dependent reversal of activated-LX2 cells was evidenced by a reduction in α-smooth muscle actin (α-SMA) and collagen secretion, and an increase in activity of matrix metalloproteases. Mechanistically, 3-BrPA-dependent antifibrotic effects involved down-regulation of the mitochondrial metabolic enzyme, ATP5E, and up-regulation of glycolysis, as evident by elevated levels of lactate dehydrogenase, lactate production and its transporter, MCT4. Finally, the antifibrotic effects of 3-BrPA were validated in vivo in a mouse model of carbon tetrachloride-induced liver fibrosis. Results from histopathology & histochemical staining for collagen and α-SMA substantiated that 3-BrPA promotes antifibrotic effects in vivo. Taken together, our data indicate that sublethal, metronomic treatment with 3-BrPA blocks the progression of liver fibrosis suggesting its potential as a novel therapeutic for treating liver fibrosis.
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Affiliation(s)
- Swathi Karthikeyan
- Division of Interventional Radiology, Russell H. Morgan Department of Radiology & Radiological Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - James J Potter
- Division of Gastroenterology and Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Jean-Francois Geschwind
- Division of Interventional Radiology, Russell H. Morgan Department of Radiology & Radiological Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
| | - Surojit Sur
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA
| | - James P Hamilton
- Division of Gastroenterology and Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Bert Vogelstein
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA
| | - Kenneth W Kinzler
- The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA
| | - Esteban Mezey
- Division of Gastroenterology and Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Shanmugasundaram Ganapathy-Kanniappan
- Division of Interventional Radiology, Russell H. Morgan Department of Radiology & Radiological Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
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Arriazu E, Ruiz de Galarreta M, Cubero FJ, Varela-Rey M, Pérez de Obanos MP, Leung TM, Lopategi A, Benedicto A, Abraham-Enachescu I, Nieto N. Extracellular matrix and liver disease. Antioxid Redox Signal 2014; 21:1078-97. [PMID: 24219114 PMCID: PMC4123471 DOI: 10.1089/ars.2013.5697] [Citation(s) in RCA: 113] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
SIGNIFICANCE The extracellular matrix (ECM) is a dynamic microenvironment that undergoes continuous remodeling, particularly during injury and wound healing. Chronic liver injury of many different etiologies such as viral hepatitis, alcohol abuse, drug-induced liver injury, obesity and insulin resistance, metabolic disorders, and autoimmune disease is characterized by excessive deposition of ECM proteins in response to persistent liver damage. CRITICAL ISSUES This review describes the main collagenous and noncollagenous components from the ECM that play a significant role in pathological matrix deposition during liver disease. We define how increased myofibroblasts (MF) from different origins are at the forefront of liver fibrosis and how liver cell-specific regulation of the complex scarring process occurs. RECENT ADVANCES Particular attention is paid to the role of cytokines, growth factors, reactive oxygen species, and newly identified matricellular proteins in the regulation of fibrillar type I collagen, a field to which our laboratory has significantly contributed over the years. We compile data from recent literature on the potential mechanisms driving fibrosis resolution such as MF' apoptosis, senescence, and reversal to quiescence. FUTURE DIRECTIONS We conclude with a brief description of how epigenetics, an evolving field, can regulate the behavior of MF and of how new "omics" tools may advance our understanding of the mechanisms by which the fibrogenic response to liver injury occurs.
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Affiliation(s)
- Elena Arriazu
- 1 Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine , New York, New York
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Potter JJ, Liu X, Koteish A, Mezey E. 1,25-dihydroxyvitamin D3 and its nuclear receptor repress human α1 (I) collagen expression and type I collagen formation. Liver Int 2013; 33:677-86. [PMID: 23413886 PMCID: PMC3707129 DOI: 10.1111/liv.12122] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/12/2012] [Accepted: 12/24/2012] [Indexed: 12/12/2022]
Abstract
BACKGROUND Vitamin D deficiency is common in chronic liver disease particularly in those with severe liver fibrosis. AIMS To determine the effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3 ) on the human α(1) (I) collagen promoter and collagen formation by human stellate LX-2 cells and the mechanism of the effect of the vitamin D receptor (VDR) on the promoter. METHODS Type I collagen was assessed by measurements of collagen mRNA and collagen protein and by transfection experiments. Binding of VDR to the α(1) (I) collagen promoter was determined by EMSA and ChIP assays. RESULTS 1,25-(OH)2 D3 decreased human α(1) (I) collagen mRNA and protein and the secretion of type I collagen by stellate cells after exposure to TGFβ1. Furthermore, 1,25-(OH)2 D3 inhibited TGFβ1-induced activation of the α(1) (I) collagen promoter in transfected LX-2 cells. The effect of 1,25-(OH)2 D3 is mediated by the VDR, which binds at a proximal Sp1 site and also at a newly identified distal site on the collagen promoter. A VDR expression vector reduced the activities of the collagen promoter in transfected LX-2 cells. CONCLUSIONS 1,25-(OH)2 D3 inhibits type I collagen formation in human stellate cells. The effect of 1,25-(OH)2 D3 is mediated by its receptor which binds at a proximal Sp1.1 site and at a newly identified distal site on the collagen promoter. Correction of vitamin D deficiency in patients with chronic liver disease is a potential therapy to inhibit progression of fibrosis.
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Affiliation(s)
- James J Potter
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
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Zhu J, Fan JR, Pan L, Huang H, Xiao MB, Jiang F, Lu CH. Correlation of nuclear factor κB expression with α-SMA and collagen Ⅲ expression in hepatic fibrosis in rats. Shijie Huaren Xiaohua Zazhi 2012; 20:2081-2085. [DOI: 10.11569/wcjd.v20.i22.2081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the correlation of expression of nuclear factor κB with that of α-smooth muscle actin (α-SMA) and collagen Ⅲ in hepatic fibrosis in rats.
METHODS: Thirty-two male SD rats of SPF grade were divided randomly into control group and model group. Hepatic fibrosis was induced in rats by injecting carbon tetrachloride. The mRNA and protein expression of NF-κB, α-SMA, and collagen Ⅲ was examined using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. The correlation of NF-κB expression with α-SMA and collagen Ⅲ expression was then analyzed.
RESULTS: NF-κB, α-SMA and collagen Ⅲ were lowly expressed in normal liver tissue. After injection of carbon tetrachloride, the expression of NF-κB, α-SMA and collagen Ⅲ mRNAs and proteins began to increase at week 2 and was significantly higher at weeks 4 and 6 (both P < 0.05), showing a gradually rising trend. There was a positive correlation between the expression of NF-κB and that of α-SMA and collagen Ⅲ (both P < 0.05).
CONCLUSION: NF-κB plays an important role in the pathogenesis of hepatic fibrosis.
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Lu B, Yu L, Li S, Si S, Zeng Y. Alleviation of CCl4-induced cirrhosis in rats by tetramethylpyrazine is associated with downregulation of leptin and TGF-β1 pathway. Drug Chem Toxicol 2010; 33:310-5. [DOI: 10.3109/01480540903418504] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
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Sysa P, Potter JJ, Liu X, Mezey E. Transforming growth factor-beta1 up-regulation of human alpha(1)(I) collagen is mediated by Sp1 and Smad2 transacting factors. DNA Cell Biol 2009; 28:425-34. [PMID: 19558215 DOI: 10.1089/dna.2009.0884] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Hepatic fibrosis results from excessive deposition of type I collagen. The roles of Smads in mediating the effect of transforming growth factor-beta1 (TGFbeta1) on activation of the alpha(1)(I) collagen promoter were determined. Smads bind in association with Sp1 to the CC(GG)-rich TGFbeta1 responsive element of the promoter that lacks the classical Smad recognition element, and enhance binding of Sp1. In transfection experiments, TGFbeta1 activated a proximal promoter, but not promoters mutated at sites that prevented Sp1 binding. Sp1 alone or the combination of Smad2 and Smad4 activated the promoter in transfected human LX-2 stellate cells. Sp1 or Smad2 knockdowns with siRNAs prevented the effect of TGFbeta1 in enhancing the promoter. In conclusion, this study shows that Smads bind in association with Sp1 to the CC(GG)-rich TGFbeta1 responsive element of the human alpha(1)(I) collagen promoter that lacks the classical Smad recognition element, thus enhancing the binding of Sp1 and in this manner activating the collagen promoter.
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Affiliation(s)
- Polina Sysa
- Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2195, USA
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Elinav E, Ali M, Bruck R, Brazowski E, Phillips A, Shapira Y, Katz M, Solomon G, Halpern Z, Gertler A. Competitive inhibition of leptin signaling results in amelioration of liver fibrosis through modulation of stellate cell function. Hepatology 2009; 49:278-86. [PMID: 19065677 DOI: 10.1002/hep.22584] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
UNLABELLED Leptin signaling is involved in T-cell polarization and is required for profibrotic function of hepatic stellate cells (HSCs). Leptin-deficient ob/ob mice do not develop liver fibrosis despite the presence of severe long-standing steatohepatitis. Here, we blocked leptin signaling with our recently generated mouse leptin antagonist (MLA), and examined the effects on chronic liver fibrosis in vivo using the chronic thioacetamide (TAA) fibrosis model, and in vitro using freshly-isolated primary HSCs. In the chronic TAA fibrosis model, leptin administration was associated with significantly enhanced liver disease and a 100% 5-week to 8-week mortality rate, while administration or coadministration of MLA markedly improved survival, attenuated liver fibrosis, and reduced interferon gamma (IFN-gamma) levels. No significant changes in weight, serum cholesterol, or triglycerides were noted. In vitro administration of rat leptin antagonist (RLA), either alone or with leptin, to rat primary HSCs reduced leptin-stimulated effects such as increased expression of alpha-smooth muscle actin (alpha-SMA), and activation of alpha1 procollagen promoter. CONCLUSION Inhibition of leptin-enhanced hepatic fibrosis may hold promise as a future antifibrotic therapeutic modality.
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Affiliation(s)
- Eran Elinav
- Institute for Gastroenterology and Liver Disease, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
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Kümpers P, Gueler F, Rong S, Mengel M, Tossidou I, Peters I, Haller H, Schiffer M. Leptin is a coactivator of TGF-beta in unilateral ureteral obstructive kidney disease. Am J Physiol Renal Physiol 2007; 293:F1355-62. [PMID: 17686962 DOI: 10.1152/ajprenal.00003.2007] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Progressive tubulointerstitial fibrosis is the common end point leading to end-stage renal disease in experimental and clinical settings. Since the peptide hormone leptin is involved not only in the regulation of obesity but also in the regulation of inflammation and fibrosis, we tested the hypothesis whether leptin deficiency has an impact on tubulointerstitial fibrosis in mice. Leptin-deficient (ob/ob) and leptin receptor-deficient mice (db/db) were exposed to 14 days of unilateral ureteral obstruction (UUO). The degree of fibrosis and inflammation was compared with that in sham-operated mice by performing immunohistochemistry, quantitative PCR, and Western blotting. We found that tubulointerstitial fibrosis was significantly reduced in the obstructed kidneys of ob/ob compared with db/db mice or control mice. Detailed analysis of infiltrating inflammatory cells by immunohistochemistry revealed a significant reduction of CD4(+) cells at 14 days after UUO in both ob/ob and db/db mice. In contrast, we could not detect significant differences in CD8(+) cells and macrophage content. Transforming growth factor (TGF)-beta mRNA levels, TGF-beta-induced Smad-2/3 activation, and the upregulation of downstream target genes were significantly reduced in ob/ob mice. In addition, we demonstrated that leptin could enhance TGF-beta signaling in normal rat kidney fibroblasts in vitro. We conclude that leptin can serve as a cofactor of TGF-beta activation and thus plays an important role in renal tubulointerstitial fibrosis. Therefore, selective blockade of the leptin axis might provide a therapeutic possibility to prevent or delay fibrotic kidney disease.
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Affiliation(s)
- Philipp Kümpers
- Department of Nephrology, Hannover Medical School, Hannover, Germany
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N/A, 刘 清. N/A. Shijie Huaren Xiaohua Zazhi 2005; 13:1801-1803. [DOI: 10.11569/wcjd.v13.i15.1801] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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Mao YM, Zeng MD, Lu LG, Wan MB, Li CZ, Chen CW, Fu QC, Wang JY, She WM, Cai X, Ye J, Zhou XQ, Wang H, Wu SM, Tang MF, Zhu JS, Chen WX, Zhang HQ. Capsule oxymatrine in treatment of hepatic fibrosis due to chronic viral hepatitis: A randomized, double blind, placebo-controlled, multicenter clinical study. World J Gastroenterol 2004; 10:3269-73. [PMID: 15484298 PMCID: PMC4572293 DOI: 10.3748/wjg.v10.i22.3269] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To evaluate the efficacy and safety of oxymatrine capsule in treatment of hepatic fibrosis in patients with chronic viral hepatitis.
METHODS: It was a randomized, double blind, placebo-controlled, multicenter clinical study. One hundred and forty-four patients were divided into oxymatrine capsule group(group A) and placebo group (group B).The course was 52 wk. Patients were visited once every 12 wk and the last visit was at 12 wk after cessation of the treatment. All patients had liver biopsy before treatment. part of them had a second biopsy at the end of therapy. Clinical symptoms, liver function test, serum markers of hepatic fibrosis were tested. Ultrasound evaluation was performed before, during and at the end of therapy.
RESULTS: One hundred and forty-four patients enrolled in the study. Of them 132 patients completed the study according to the protocol,49 patients had liver biopsy twice (25 patients in group A and 24 in group B). At the end of therapy, significant improvements in hepatic fibrosis and inflammatory activity based on Semi-quantitative scoring system (SSS) were achieved in group A. The total effective rate of the treatment was 48.00%, much higher than that of 4.17% in group B (P < 0.05). Significant improvement in serum markers of hepatic fibrosis such as hyaluronic acid (HA) and type III procollagenic peptide (P III P) in group A was seen (P < 0.05). The total effective rate of serum markers at the end of therapy in group A was 68.19%, much higher than that of 34.85% in group B (P < 0.05). The total effective rate of noninvasive markers at the end of therapy in group A was 66.67%, much higher than that of 30.30% in group B (P < 0.05). The rate of adverse events was similar in two groups.
CONCLUSION: Oxymatrine capsule is effective and safe in treatment of hepatic fibrosis due to chronic viral hepatitis.
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Affiliation(s)
- Yi-Min Mao
- Shanghai Institute of Digestive Disease, Renji Hospital, Shanghai Second Medical University, Shanghai 200001, China.
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