A New Murine Liver Fibrosis Model Induced by Polyhexamethylene Guanidine-Phosphate

Kim Minjeong,Hur Sumin,김광휘,Cho Yejin,Kim Keunyoung,Kim Ha Ryong,남기택,Lim Kyung-Min 한국응용약물학회 2022 Biomolecules & Therapeutics(구 응용약물학회지) Vol.30 No.2

Liver fibrosis is part of the wound healing process to help the liver recover from the injuries caused by various liver-damaging insults. However, liver fibrosis often progresses to life-threatening cirrhosis and hepatocellular carcinoma. To overcome the limitations of current in vivo liver fibrosis models for studying the pathophysiology of liver fibrosis and establishing effective treatment strategies, we developed a new mouse model of liver fibrosis using polyhexamethylene guanidine phosphate (PHMG-p), a humidifier sterilizer known to induce lung fibrosis in humans. Male C57/BL6 mice were intraperitoneally injected with PHMG-p (0.03% and 0.1%) twice a week for 5 weeks. Subsequently, liver tissues were examined histologically and RNA-sequencing was performed to evaluate the expression of key genes and pathways affected by PHMG-p. PHMG-p injection resulted in body weight loss of ~15% and worsening of physical condition. Necropsy revealed diffuse fibrotic lesions in the liver with no effect on the lungs. Histology, collagen staining, immunohistochemistry for smooth muscle actin and collagen, and polymerase chain reaction analysis of fibrotic genes revealed that PHMG-p induced liver fibrosis in the peri-central, peri-portal, and capsule regions. RNA-sequencing revealed that PHMG-p affected several pathways associated with human liver fibrosis, especially with upregulation of lumican and IRAK3, and downregulation of GSTp1 and GSTp2, which are closely involved in liver fibrosis pathogenesis. Collectively we demonstrated that the PHMG-p-induced liver fibrosis model can be employed to study human liver fibrosis.

간 섬유화 진단을 위한 Liver Shear Wave Elastography의 유용성

이병춘(Byeong Choon Lee),김현진(Hyeon Jin Kim),정양화(Yang Hwa Chung),최준호(Jun Ho Choi),이원홍(Won Hong Lee),차현정(Hyeon Jeong Cha) 대한초음파의료영상학회 2012 대한초음파의료영상학회지 Vol.3 No.1

목적 : 간 섬유화(Liver fibrosis) 진단에 있어 Liver Shear wave elastography와 간 조직생검의 결과를 비교하여 Liver Shear wave elastography의 임상적 유용성을 알아보고자 한다. 대상 및 방법 : 2011년 9월부터 2012년 2월까지 본원에 내원한 B형 또는 C형 간염 보균자 54명을 대상으로 Liver Shear wave elastogrphy와 간 조직생검을 같이 시행하였다. 간 조직생검의 섬유화 정도는 METAVIR 기준의 간 섬유화 단계에 따라 분류하였다. Liver Shear wave elastography 수치는 3회씩 측정하여 평균치를 구한 후, Ultrasound in Medicine & Biology 2011에 나와있는 Liver stiffness optimal cutoff value 값과 비교 분석하였다. 결과 : 전체 대상 54명 중 간 조직생검으로 나온 간 섬유화 정도는 F0~F1은 20명(37%), F2, F3는 각각 22명 (41%), 7명(13%)이었고, F4는 5명(9%)이었다. 간 섬유화 단계에 따라 간 탄성도 수치를 Optimal cutoff value값과 비교분석한 결과, F0~F1:95%(19명), F2:90%(20명), F3:85%(6명), F4:65%(3명)가 optimal cutoff value값에 일치함을 확인하였다. 결론 : Liver Shear wave elastogrphy가 간 섬유화를 진단하는데 있어, F0~F1, F2의 간 섬유화 단계에서 90% 이상 일치율을 보였으며, 이는 간 섬유화 조기 진단에 유용성이 있을 것으로 사료된다. Purpose : The purpose is to determine clinical usefulness of liver Share wave elastography in diagnosing liver fibrosis by comparing test results of liver Share wave elastography and liver biopsy. Subjects and Methods : tests of liver Share wave elastography and liver biopsy have been done on 54 inpatients who carry hepatitis Type B and Type C between the period of September of 2011 and February of 2012. The degrees of liver biopsy was classified according to the levels of liver fibrosis based on METAVIR. The rates of Liver Shear wave elastography were measured three times and their average was compared and analyzed with liver stiffness optimal cutoff value published on the Ultrasound in Medicine & Biology 2011. Results : Of all the 54 subjects, 20, 37% of them, were in the range of F0-F1 in liver fibrosis through liver biopsy. Those in the range of F2 and F3 were 22, 41% and 7, 13% respectively. The results of comparing rates of liver stiffness with Optimal cutoff value according to the liver fibrosis showed that 19 subjects, 95%, were in the range of F0-F1, 20 subjects, 90% in F2, 6 subjects, 85% in F3 and 3 subjects, 65% in F4 turned out to fit the optimal cutoff value. Conclusion : Liver Shear wave elastography tests, diagnosing the liver fibrosis, matched more than 90% in the levels of F0~F1 and F2 liver fibrosis. This is considered to be useufl in early diagnosis of liver fibrosis.

종설 : FibroScan(R)을 이용한 간탄력도 검사

한광협 ( Kwang Hyub Han ),김승업 ( Seung Up Kim ) 대한내과학회 2008 대한내과학회지 Vol.74 No.5

Progressive liver fibrosis is a similar feature of all chronic liver diseases and eventually develops liver cirrhosis. The prognosis and treatment plans of chronic liver diseases depend strongly on the degree of liver fibrosis. These facts raise clinical interests in quantifying liver fibrosis. Although liver biopsy has been the gold standard for assessment of liver fibrosis, it has some technical limitations and risks. Accordingly, an increasing need for alternative non-invasive method to quantify liver fibrosis has been a major challenge that has stimulated search for new non-invasive methods. Such methods for diagnosing liver fibrosis have progressed significantly over the last few years notably with the appearance of several serological markers which have been reported to predict the presence of significant fibrosis or cirrhosis in patients with chronic liver disease with considerable accuracy. However, complicated calculation, cost problems, and influences of extrahepatic conditions make it less accessible to clinicians. Recently, liver stiffness measurement using FibroScan(R) is emerging as a new diagnostic method for liver fibrosis. It is totally non-invasive and reproducible and gives an immediate result without intra- and inter-observer variability. Its clinical use in comparison with liver biopsy and several available serologic markers is now intensively being investigated. Here, we review the currently available data on FibroScan(R).(Korean J Med 74:463-471, 2008)

Original Article : Magnetization-tagged MRI is a simple method for predicting liver fibrosis

( Kyung Eun Kim ),( Mi Suk Park ),( Sohae Chung ),( Chansik An ),( Leon Axel ),( Rakhmonova Gulbahor Ergashovna ) 대한간학회 2016 Clinical and Molecular Hepatology(대한간학회지) Vol.22 No.1

Background/Aims: To assess the usefulness of magnetization-tagged magnetic resonance imaging (MRI) in quantifying cardiac-induced liver motion and deformation in order to predict liver fibrosis. Methods: This retrospective study included 85 patients who underwent liver MRI including magnetization-tagged sequences from April 2010 to August 2010. Tagged images were acquired in three coronal and three sagittal planes encompassing both the liver and heart. A Gabor filter bank was used to measure the maximum value of displacement (MaxDisp) and the maximum and minimum values of principal strains (MaxP1 and MinP2, respectively). Patients were divided into three groups (no fibrosis, mild-to-moderate fibrosis, and significant fibrosis) based on their aspartate-aminotransferase-to-platelet ratio index (APRI) score. Group comparisons were made using ANOVA tests. Results: The patients were divided into three groups according to APRI scores: no fibrosis (≤0.5; n=41), moderate fibrosis (0.5-1.5; n=23), and significant fibrosis (>1.5; n=21). The values of MaxDisp were 2.9±0.9 (mean±SD), 2.3±0.7, and 2.1±0.6 in the no fibrosis, moderate fibrosis, and significant fibrosis groups, respectively (P<0.001); the corresponding values of MaxP1 were 0.05±0.2, 0.04±0.02, and 0.03±0.01, respectively (P=0.002), while those of MinP2 were -0.07±0.02, -0.05±0.02, and -0.04±0.01, respectively (P<0.001). Conclusions: Tagged MRI to quantify cardiac-induced liver motion can be easily incorporated in routine liver MRI and may represent a helpful complementary tool in the diagnosis of early liver fibrosis. (Clin Mol Hepatol 2016;22:140-145)

만성 간질환 환자에서 간 섬유화 평가에 대한 간 탄력도 측정의 유용성

김은선 ( Eun Sun Kim ),서연석 ( Yeon Seok Seo ),이광균 ( Kwang Gyun Lee ),박상훈 ( Sanghoon Park ),권용대 ( Yong Dae Kwon ),금보라 ( Bora Keum ),김용식 ( Yong Sik Kim ),진윤태 ( Yoon Tae Jeen ),전훈재 ( Hoon Jai Chun ),김창덕 ( Ch 대한내과학회 2008 대한내과학회지 Vol.74 No.3

목적: 만성간질환 환자에서 섬유화 진행 정도의 평가는 환자의 치료 및 예후를 결정하는데 매우 중요하다. 섬유화 평가를 위해 간 조직 생검이 가장 좋은 검사법으로 알려져 있으나 비용이 많이 들고 침습적인 검사라는 점 등 몇 가지 문제점이 있어 최근에는 비 침습적 방법을 통한 간 섬유화 측정법들이 시도되고 있다. 본 연구는 이러한 비 침습적 검사법의 하나인 Fibroscan을 통한 liver stiffness measurement(LSM)가 만성간질환 환자에서의 간 섬유화 정도 평가에 유용한가를 평가하기 위해 시행하였다. 방법: 만성간질환으로 간 조직생검을 시행한 93명의 환자를 대상으로 하였다. 간 조직생검 시행 당일 또는 다음날 Fibroscan을 통해 LSM을 측정하였다. 생검 조직의 섬유화 정도는 METAVIR 기준의 간 섬유화 병기에 따라 분류하였다. LSM 결과의 유용성 및 섬유화 정도 평가에 대한 optimal cutoff value를 결정하기 위해 receiver-operating characteristics(ROC) curve 분석을 시행하였다. 결과: 만성간질환으로 간 조직생검을 시행 받은 85명 환자의 연령은 40±13세(중앙값, 40세)였으며 남자가 62명(66.6%)이었다. 만성간질환의 원인은 다음과 같다. 만성 B형 간염, 56명(65.9%) 만성 C형 간염, 19명(22.4%) 지방간질환, 4명(4.7); 자가면역성간염, 3명(3.5%) 기타, 2명(2.4%). 간 조직 생검 결과 F0-1, F2, F3 및 F4에 해당하는 환자는 각각 21명(24.7%), 27명(31.8%), 26명(30.6%) 및 11명(12.9%)이었다. 간조직 생검을 통한 간 섬유화 진행단계는 LSM 결과와 유의한 연관성을 보였다(Kendall`s correlation coefficient: 0.583;p<0.001). 간 조직 생검에서 F2 이상, F3 이상 및 F4 이상에 대한 LSM의 area under ROC curve는 각각 0.871 (95% CI, 0.787-0.956), 0.874 (95% CI, 0.800-0.948) 및 0.894 (95% CI, 0.817-0.970)였다. F2 이상, F3 이상 및 F4 이상에 대한 optimal cutoff value는 각각 6.9, 11.9 및 14.15 kPa였다. 결론: Fibroscan을 통한 LSM 측정은 만성간질환 환자에서 간의 섬유화 정도를 평가할 수 있는 유용한 비 침습적인 방법으로 생각된다. Background/Aims: Accurate assessment of liver fibrosis is very important for predicting the prognosis of patients with chronic liver diseases. Liver biopsy is still considered the gold-standard for assessing liver fibrosis. However, it is an invasive procedure with several limitations such as its questionable outcomes. Recent studies have suggested that liver stiffness measurement (LSM) using Fibroscan is noninvasive and useful for assessing liver fibrosis. This study was performed to evaluate the efficacy of LSM for evaluating liver fibrosis in patients with chronic liver diseases. Methods: We prospectively enrolled 93 patients with chronic liver diseases, as confirmed by liver biopsy. The patients underwent liver biopsy and LSM. The METAVIR liver fibrosis stages of the biopsy specimens were assessed by an experienced pathologist. LSM was performed by Fibroscan. The efficacy of LSM and the optimal cutoff values for assessment of the fibrosis stage were determined by a receiver-operating characteristics (ROC) curve analysis. Results: LSM was well correlated with the fibrosis stage (Kendall correlation coefficient: 0.58; p<0.001). The areas under the ROC curves were 0.871 (95% CI, 0.715-0.924) for the patients with significant fibrosis (F≥2), 0.874 (0.761-0.929) for the patients with severe fibrosis (F≥3) and 0.894 (0.792-0.956) for the patients with cirrhosis (F=4). The optimal LSM cutoff values for F ≥2, F ≥3 and F=4 were 6.9, 11.75 and 14.5 kPa, respectively. Conclusions: LSM was a simple, effective method for assessing liver fibrosis in patients with chronic liver diseases. Its use for the follow up and management of these patients could be of great interest and so further investigation is required. (Korean J Med 74:264-270, 2008)

Thioacetamide로 유도된 간섬유화 모델에서 생간건비탕(生肝健脾湯)의 보호 효과

최정원,정성미,신미래,정다운,노성수,Choi, Jeong Won,Chung, Sung Mi,Shin, Mi-Rae,Jeong, Da un,Roh, Seong-Soo 대한본초학회 2022 大韓本草學會誌 Vol.37 No.6

Objective : In modern society, liver diseases such as liver fibrosis are on the rise as inflammation and wound healing processes of the liver are repeated due to factors such as drinking, smoking, and stress. This study was conducted to evaluate the effect of Saenggangeonbi-tang (SGGBT) on thioacetamide (TAA)-induced liver fibrosis. Methods : The mice were divided into 4 groups for examination (n=6): Normal group (Nor), distilled water-treated liver fibrosis mice (Con), silymarin 50 mg/kg-treated liver fibrosis mice (Sily), SGGBT 200 mg/kg-treated liver fibrosis mice (S200). Liver fibrosis was established in the mice via TAA for 8 weeks (1 week 100 mg/kg, 2,3 weeks 200 mg/kg, 4-8 weeks 400 mg/kg, three times a week, intraperitoneal injection) and they were administered silymarin and SGGBT (every day, oral administration) with the TAA. Results : SGGBT significantly decreased the levels of aspartate aminotransferase, alanine aminotransferanse, ammonia, and myeloperoxidase in serum increased by liver fibrosis. As a result of confirming H&E and MT staining, it was confirmed that SGGBT reduced damage and inflammatory cell infiltration in liver tissue, and alleviated changes in collagen fiber deposition and histological fibrosis. Also, it was confirmed through PAS staining that it reduced glycogen deposition in liver tissue. In addition, SGGBT significantly decreased the NADPH oxidases as well as significantly modulated the expression of MMP-2 and TIMP-2. Conclusions : These results suggest that SGGBT regulates the expression of MMP/TIMP protein through inhibition of oxidative stress and alleviates liver fibrosis by reducing collagen and glycogen deposition in liver tissue.

Protective effect of Saenggangeonbi-tang on liver fibrosis induced by thioacetamide

( Chung Sung Mi ) 대구한의대학교 제한동의학술원 2022 제한동의학술원논문집 Vol.20 No.1

Objective : In modern society, liver diseases such as liver fibrosis are on the rise as inflammation and wound healing processes of the liver are repeated due to factors such as drinking, smoking, and stress. This study was conducted to evaluate the effect of (SGGBT) on thioacetamide (TAA)-induced liver fibrosis. Methods : The mice were divided into 4 groups for examination (n=6): Normal group (Nor), distilled water-treated liver fibrosis mice (Con), silymarin 50 mg/kg-treated liver fibrosis mice (Sily), SGGBT 200 mg/kg-treated liver fibrosis mice (S200). Liver fibrosis was established in the mice via TAA for 8 weeks (1 week 100 mg/kg, 2,3 weeks 200 mg/kg, 4-8 weeks 400 mg/kg, three times a week, intraperitoneal injection) and they were administered silymarin and SGGBT (every day, oral administration) with the TAA. Results : SGGBT significantly decreased the levels of aspartate aminotransferase, alanine aminotransferanse, ammonia, and myeloperoxidase in serum increased by liver fibrosis. As a result of confirming H&E and MT staining, it was confirmed that SGGBT reduced damage and inflammatory cell infiltration in liver tissue, and alleviated changes in collagen fiber deposition and histological fibrosis. Also, it was confirmed through PAS staining that it reduced glycogen deposition in liver tissue. In addition, SGGBT significantly decreased the NADPH oxidases as well as significantly modulated the expression of MMP-2 and TIMP-2. Conclusions : These results suggest that SGGBT regulates the expression of MMP/TIMP protein through inhibition of oxidative stress and alleviates liver fibrosis by reducing collagen and glycogen deposition in liver tissue.

Establishment of Two Liver Fibrosis Models to Examine Endothelial Progenitor Cell Kinetics

( Katsuya Shirakura ),( Sang Mo Kwon ),( Haruchika Masuda ),( Syotaro Obi ),( Rie Ito ),( Tomoko Shizuno ),( Yusuke Kurihara ),( Tetsuya Mine ),( Takayuki Asahara ) 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.12

Chronic inflammatory liver damage frequently induces irreversible liver fibrosis that eventually results in liver cirrhosis. Thus far, liver transplantation has been used as a non-optional treatment for patients with severe liver cirrhosis, although there are several drawbacks, including the shortage of donors and invasiveness of the operation. Bone marrow or peripheral blood derived stem cells, including endothelial progenitor cells (EPCs), have recently been used to treat patients with liver cirrhosis as a means of liver regeneration therapy, however, there have been very few reports on EPC kinetics during the development of liver fibrosis. To investigate the relationship between EPC kinetics and liver fibrosis, we have tried two murine models of inflammatory liver fibrosis, one created by injecting carbon tetrachloride (CCl4) intraperitoneally and the other created by injecting CCl4 subcutaneously. Examination of hematoxylin-eosin and azan stained sections of the animals` liver confirmed that both models exhibited the typical characteristics of liver fibrosis, including bridging fibrosis and pseudolobule formation, and splenomegaly was also observed. In addition, the blood levels of liver transaminases, alkaline phosphatase, and hyaluronic acid increased in the CCl4- injected mice, but not in the control mice. Importantly, EPC colony-forming assays showed a significant decrease in CFU-EPC numbers in both liver fibrosis models, suggesting that functional EPC bioactivity in the bone marrow may be dramatically down-regulated in inflammatory liver fibrosis. In conclusion, our models provide information on EPC biology in chronic inflammatory liver disease that induces liver fibrosis.

Novel Therapeutic Tool for Liver Disease through Direct Conversion Hepatocyte Transplantation

( Su Hyun Park ),( Seon In Hwang ),( Seo Yeon Hwang ),( So Hee Kang ),( Sera Yang ),( Jonghwa Kim ),( Wonseok Kang ),( Kyun-hwan Kim ),( Dong Wook Han ),( Yong-han Paik ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: Until now there was no anti-fibrotic therapy available for liver cirrhosis. Stem cell therapies have been studied for the treatment of liver fibrosis. However, use of embryonic stem cell or induced pluripotent stem cell (iPSC) has limitations such as ethical concern or malignancy potential. Induced hepatocytes (iHEPs) generated by direct reprogramming technology may overcome these limitations. In this study we investigated the effect of iHEPs on acute liver injury and liver fibrosis induced by CCl4 intraperitoneal injection in mice. Methods: iHEPs were generated from mouse embryonic fibroblasts (MEFs) by direct reprogramming. Acute liver injury was induced by CCl4 intraperitoneal injection and GFP-labeled iHEPs were transplanted after 24h of CCl4 injection. Liver fibrosis was induced in BALB/c nude mice by intraperitoneal injection of CCl4 for 10 weeks. GFP-labeled iHEPs (1x106 cells in 100μl DMEM) were transplanted at week 8 by intrasplenic injection. Liver injury was assessed by serum ALT and AST measurement. Liver histology and fibrosis was assessed by H&E staining and Sirius Red staining. Results: In acute liver injury model, CCl4 induced AST and ALT elevation which was significantly reduced by transplantation of iHEPs (p<0.01). GFP- and albumin-expression were co-localized nearby the damaged portal vein area, indicating successful migration of transplanted iHEPs to the injured hepatic regions. GFP expression was detectable up to 72h post-transplantation by Western blot, suggesting persistence of transplanted iHEPs during the course of acute liver injury. In liver fibrosis model, liver injury was diminished by transplantation of iHEPs. Sirius Red staining revealed a significant reduction of fibrosis area in iHEP-transplantation group compared to control group (p<0.0001). Conclusions: We confirmed that iHEPs generated by direct reprogramming migrated to the liver after intrasplenic transplantation. Transplantation of iHEPs significantly attenuated acute liver injury and liver fibrosis induced by CCl4 injections. These data suggest that iHEPs may serve as a novel therapeutic strategy for treatment of liver fibrosis.

간섬유화

한광협 ( Kwang Hyub Han ),김승업 ( Seung Up Kim ) 대한간학회 2010 Clinical and Molecular Hepatology(대한간학회지) Vol.16 No.2(S)

Liver fibrosis is the results of chronic injury and a similar feature of all chronic liver diseases. Beyond being a marker of injury, it appears to play a direct role in the pathogenesis of hepatocellular dysfunction and portal hypertension. Furthermore, the prognosis and treatment plans of chronic liver diseases strongly depend on the degree of liver fibrosis. Thus, from a clinical management viewpoint, accurately assessing the extent and progression of fibrosis is important and clinical interests are being raised in quantifying liver fibrosis. Although liver biopsy has been the gold standard for assessment of liver fibrosis, it has some technical limitations and risks. Accordingly, an increasing need for alternative noninvasive method to quantify liver fibrosis has been a major challenge that has stimulated search for new noninvasive methods. Such methods for diagnosing liver fibrosis have progressed significantly over the last few decades notably with the appearance of several serological markers, which have been reported to predict the presence of significant fibrosis or cirrhosis in patients with chronic liver disease with considerable accuracy. However, complicated calculation and influences of extrahepatic conditions make it less accessible to clinicians. Recently, transient elastography using FibroScan(R) is emerging as a new diagnostic method for liver fibrosis. It is totally noninvasive and reproducible and gives an immediate result with low intra- and inter-observer variability. Here, we review the currently available data on transient elastography for assessing liver fibrosis.