Histological and Physiological Studies of the Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Bleomycin Induced Lung Fibrosis in Adult Albino Rats

Zakaria Dina Mohamed,Zahran Noha Mahmoud,Arafa Samia Abdel Aziz,Mehanna Radwa Ali,Abdel-Moneim Rehab Ahmed 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.1

Background: Lung fibrosis is considered as an end stage for many lung diseases including lung inflammatory disease, autoimmune diseases and malignancy. There are limited therapeutic options with bad prognostic outcome. The aim of this study was to explore the effect of mesenchymal stem cells (MSCs) derived from bone marrow on Bleomycin (BLM) induced lung fibrosis in albino rats. Methods: 30 adult female albino rats were distributed randomly into 4 groups; negative control group, Bleomycin induced lung fibrosis group, lung fibrosis treated with bone marrow-MSCs (BM-MSCs) and lung fibrosis treated with cell free media. Lung fibrosis was induced with a single dose of intratracheal instillation of BLM. BM-MSCs or cell free media were injected intravenously 28 days after induction and rats were sacrificed after another 28 days for assessment. Minute respiratory volume (MRV), forced vital capacity (FVC) and forced expiratory volume 1 (FEV1) were recorded using spirometer (Power lab data acquisition system). Histological assessment was performed by light microscopic examination of H&E, and Masson’s trichrome stained sections and was further supported by morphometric studies. In addition, electron microscopic examination to assess ultra-structural changes was done. Confocal Laser microscopy and PCR were used as tools to ensure MSCs homing in the lung. Results: Induction of lung fibrosis was confirmed by histological examination, which revealed disorganized lung architecture, thickened inter-alveolar septa due excessive collagen deposition together with inflammatory cellular infiltration. Moreover, pneumocytes depicted variable degenerative changes. Reduction in MRV, FVC and FEV1 were recorded. BM-MSCs treatment showed marked structural improvement with minimal cellular infiltration and collagen deposition and hence restored lung architecture, together with lung functions. Conclusion: MSCs are promising potential therapy for lung fibrosis that could restore the normal structure and function of BLM induced lung fibrosis. Background: Lung fibrosis is considered as an end stage for many lung diseases including lung inflammatory disease, autoimmune diseases and malignancy. There are limited therapeutic options with bad prognostic outcome. The aim of this study was to explore the effect of mesenchymal stem cells (MSCs) derived from bone marrow on Bleomycin (BLM) induced lung fibrosis in albino rats. Methods: 30 adult female albino rats were distributed randomly into 4 groups; negative control group, Bleomycin induced lung fibrosis group, lung fibrosis treated with bone marrow-MSCs (BM-MSCs) and lung fibrosis treated with cell free media. Lung fibrosis was induced with a single dose of intratracheal instillation of BLM. BM-MSCs or cell free media were injected intravenously 28 days after induction and rats were sacrificed after another 28 days for assessment. Minute respiratory volume (MRV), forced vital capacity (FVC) and forced expiratory volume 1 (FEV1) were recorded using spirometer (Power lab data acquisition system). Histological assessment was performed by light microscopic examination of H&E, and Masson’s trichrome stained sections and was further supported by morphometric studies. In addition, electron microscopic examination to assess ultra-structural changes was done. Confocal Laser microscopy and PCR were used as tools to ensure MSCs homing in the lung. Results: Induction of lung fibrosis was confirmed by histological examination, which revealed disorganized lung architecture, thickened inter-alveolar septa due excessive collagen deposition together with inflammatory cellular infiltration. Moreover, pneumocytes depicted variable degenerative changes. Reduction in MRV, FVC and FEV1 were recorded. BM-MSCs treatment showed marked structural improvement with minimal cellular infiltration and collagen deposition and hence restored lung architecture, together with lung functions. Conclusion: MSCs are promising potential therapy for lung fibrosis that could restore the normal structure and function of BLM induced lung fibrosis.

LC, Acute : PE-112 ; Combined effects of caspase inhibitor Nivocasan and lithospermate B on the inhibition of hepatic fibrosis in rats

( Do Young Kim ),( Sook In Chung ),( Weonsang Ro ),( Yong Han Paik ),( Young Nyun Park ),( Sang Hoon Ahn ),( Jung Gyu Park ),( Hee Dong Park ),( Kwan Sik Lee ),( Kwang Hyub Han ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.-

Background/Aim: To see combined anti-fibrosis effect when lithospermate B (LAB), an anti-oxidant, and Nivocasan, a caspase inhibitor, were simultaneously administered in comparison with either compound. Methods: Hepatic fibrosis was induced in SD rats by thioacetamide (TAA). In fibrosis-preventive experiment, rats were injected with TAA and fed on LAB and Nivocasan at the same time. Grouping was; Normal control (N), Normal+ LAB+ Nivocasan (NLN), TAA control (T), TAA+LAB (TL), TAA+ Nivocasan (TN), TAA+LAB+Nivocasan (TLN). In fibrosis- reversal experiment, rats were first treated with TAA, and then LAB and Nivocasan were fed. Grouping was; TAA control (F), TAA+LAB (FL), TAA+Nivocasan (FN), and TAA+LAB+ Nivocasan (FLN). Fibrotic area was evaluated quantitatively by computerized morphometry. Apoptosis was assessed using TUNEL assay, and immunohistochemical staining for malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4HNE) was performed to assess oxidative stress. Real-time quantitative PCR was used to determine expression of fibrosis-related genes. Results: The degree of hepatic fibrosis was significantly reduced in group TLN (4.9%) compared to TL (6.3%) or TN (6.7%) (p < 0.001). These results were similarly observed in fibrosis- reversal experiment. Treatment with each compound significantly decreased fibrosis-related gene expression such as type I collagen α1 (col1α1), α-SMA, and TGF-β1 (p < 0.05). Co-treat- ment with LAB and Nivocasan further reduced col1α1 expression compared with either compound in both fibrosis- preventive and reversal experiment. TUNEL assay revealed that hepatocyte apoptosis significantly decreased in group TN and TLN compared to TL (p < 0.001). Similarly, apoptosis reduced significantly in group FN and FLN compared to FL (p < 0.001). Immunohistochemistry showed decrease of MDA and 4HNE, reflecting amelioration of oxidative stress in both fibrosis- preventive and reversal experiment, when LAB or LAB+ Nivocasan was administered compared with Nivocasan alone (p < 0.05). Conclusion: Simultaneous administration of LAB and Nivocasan to suppress oxidative stress and apoptosis resulted in an enhanced effect on inhibition of hepatic fibrosis in rats.

Role of Fibroscan and APRI Score in Detection of Liver Fibrosis in Patients with Hepatitis B

( Oidov Baatarkhuu ),( Dashchirev Munkh-orshikh ),( Baasankhuu Enkhtuvshin ) 대한간학회 2020 춘·추계 학술대회 (KASL) Vol.2020 No.1

Aims: The assessment of liver fibrosis is essential for predicting the prognosis and outcome of all forms of chronic liver disease. A liver biopsy is the gold standard for the assessment of liver fibrosis, but it has its limitations which include life-threatening complications. Alternative methods of non-invasive laboratory and radiological testing for the assessment of liver fibrosis in hepatitis have evolved during the past decade and these methods may be able to overcome the limitations of liver biopsy. This study was conducted in order to asses liver fibrosis using Fibroscan and to compare these results to the AST. Platelet ratio index (APRI scores) on HBV patients. Methods: A cross-sectional study was conducted on HBV patients who underwent Fibroscan examinations between March 15, 2015 and February 30, 2017 in Happy Veritas Clinic and Diagnostic Center. Demographic data was collected including sex, age, and nationality, serum alanine aminotransferase levels (ALT 6-24 U/l), serum aspartate aminotransferase levels (13-33U/L) and platelet counts (180-320-10⁹) wre also determined. The stages of fibrosis (F0 0-7.2; F1 7.2-8.2; F2 8.2-11; F3 11-18.3 and F4 >18.3) were in kPa. The result of APRI was compared with the Fibroscan fibrosis scores. Results: The results of 228 patients were analyzed including 126(55%) males with a mean age of 42 years (SD: 9.9, range : 22-67). The males were significantly younger than the females (47 years (SD: 10.5 (range 18-72) (P<0,001). The mean stiffness score was 11:29(SD: 8.7)kPa and most patients exhibited no fibrosis (37%) and mid-moderate level (38%) of fibrosis. Thirty patients (13%) had advanced fibrosis. The mean platelet and serum ALT levels were 1.11 (SD: 1.42; range 0.12-3.7). There was a significant positive correlation between the Fibroscan results and the APRI scores (P<0,001). Similarly, there was a significant positive correlation between age and fibrosis score and a significant negative correlation between platelet count and stiffness score. Conclusions: This study has shown that the combination of Fibroscan and APRI methods providers a valuable approach for assessing liver fibrosis in patients with hepatitis. This can eliminate the need for liver biopsy in patients without clear indication.

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.

Texture Analysis of Gray-Scale Ultrasound Images for Staging of Hepatic Fibrosis

박언주,김승호,박상준,백태욱 대한영상의학회 2021 대한영상의학회지 Vol.82 No.1

Purpose To evaluate the feasibility of texture analysis of gray-scale ultrasound (US) images for staging of hepatic fibrosis. Materials and Methods Altogether, 167 patients who had undergone routine US and laboratory tests for a fibrosis-4 (FIB-4) index were included. Texture parameters were measured using a dedicated in-house software. Regions of interest were placed in five different segments (3, 5, 6, 7, 8) for each patient. The FIB-4 index was used as the reference standard for hepatic fibrosis grade. Comparisons of the texture parameters between different fibrosis groups were performed with the Student’s t-test or Mann-Whitney U-test. Diagnostic performance was evaluated by receiver operating curve analysis. Results The study population comprised of patients with no fibrosis (FIB-4 < 1.45, n = 50), mild fibrosis (1.45 ≤ FIB-4 ≤ 2.35, n = 37), moderate fibrosis (2.35 < FIB-4 ≤ 3.25, n = 27), and severe fibrosis (FIB-4 > 3.25, n = 53). Skewness in hepatic segment 5 showed a difference between patients with no fibrosis and mild fibrosis (0.2392 ± 0.3361, 0.4134 ± 0.3004, respectively, p = 0.0109). The area under the curve of skewness for discriminating patients with no fibrosis from those with mild fibrosis was 0.660 (95% confidence interval, 0.551–0.758), with an estimated accuracy, sensitivity, specificity of 64%, 87%, 48%, respectively. Conclusion A significant difference was observed regarding skewness in segment 5 between patients with no fibrosis and patients with mild fibrosis.

Role of Fibroscan and APRI Score in Detection of Liver Fibrosis in Patients with Hepatitis B

( O. Baatarkhuu ),( M. Oyun-erdene ),( D. Munkh-orshikh ),( Ts. Gerelchimeg ),( J. Amarsanaa ) 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: The assessment of liver fibrosis is essential for predicting the prognosis and outcome of all forms of chronic liver disease. A liver biopsy is the gold standard for the assessment of liver fibrosis, but it has its limitations which include life-threatening complications. Alternative methods of non-invasive laboratory and radiological testing for the assessment of liver fibrosis in hepatitis have evolved during the past decade and these methods may be able to overcome the limitations of liver biopsy. This study was conducted in order to asses liver fibrosis using Fibroscan and to compare these results to the AST. Platelet ratio index (APRI scores) on HBV patients. Methods: A cross-sectional study was conducted on HBV patients who underwent Fibroscan examinations between March 15, 2015 and February 30, 2017 in Happy Veritas Clinic and Diagnostic Center. Demographic data was collected including sex, age, and nationality, serum alanine aminotransferase levels (ALT 6-24 U/l), serum aspartate aminotransferase levels (13- 33U/L) and platelet counts (180-320-10⁹) wre also determined. The stages of fibrosis (F0 0-7.2; F1 7.2-8.2; F2 8.2-11; F3 11- 18.3 and F4 >18.3) were in kPa. The result of APRI was compared with the Fibroscan fibrosis scores. Results: The results of 228 patients were analyzed including 126(55%) males with a mean age of 42 years (SD:9.9, range :22-67). The males were significantly younger than the females (47 years (SD:10.5 ( range 18-72) (P<0,001). The mean stiffness score was 11:29(SD:8.7)kPa and most patients exhibited no fibrosis (37%) and mid-moderate level (38 %) of fibrosis. Thirty patients(13%) had advanced fibrosis. The mean platelet and serum ALT levels were 1.11 (SD:1.42; range 0.12-3.7). There was a significant positive correlation between the Fibroscan results and the APRI scores (P<0,001). Similarly, there was a significant positive correlation between age and fibrosis score and a significant negative correlation between platelet count and stiffness score. Conclusions: This study has shown that the combination of Fibroscan and APRI methods providers a valuable approach for assessing liver fibrosis in patients with hepatitis. This can eliminate the need for liver biopsy in patients without clear indication.

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)

Updates in the quantitative assessment of liver fibrosis for nonalcoholic fatty liver disease: Histological perspective

( Gwyneth Soon ),( Aileen Wee ) 대한간학회 2021 Clinical and Molecular Hepatology(대한간학회지) Vol.27 No.1

Nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) is a major cause of liver fibrosis and cirrhosis. Accurate assessment of liver fibrosis is important for predicting disease outcomes and assessing therapeutic response in clinical practice and clinical trials. Although noninvasive tests such as transient elastography and magnetic resonance elastography are preferred where possible, histological assessment of liver fibrosis via semiquantitative scoring systems remains the current gold standard. Collagen proportionate area provides more granularity by measuring the percentage of fibrosis on a continuous scale, but is limited by the absence of architectural input. Although not yet used in routine clinical practice, advances in second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) microscopy imaging show great promise in characterising architectural features of fibrosis at the individual collagen fiber level. Quantification and calculation of different detailed variables of collagen fibers can be used to establish algorithm-based quantitative fibrosis scores (e.g., qFibrosis, q-FPs), which have been validated against fibrosis stage in NAFLD. Artificial intelligence is being explored to further refine and develop quantitative fibrosis scoring methods. SHG-microscopy shows promise as the new gold standard for the quantitative measurement of liver fibrosis. This has reaffirmed the pivotal role of the liver biopsy in fibrosis assessment in NAFLD, at least for the near-future. The ability of SHG-derived algorithms to intuitively detect subtle nuances in liver fibrosis changes over a continuous scale should be employed to redress the efficacy endpoint for fibrosis in NASH clinical trials; this approach may improve the outcomes of the trials evaluating therapeutic response to antifibrotic drugs. (Clin Mol Hepatol 2021;27:44-57)

The Diagnostic Efficacy of M2BPGi for Liver Fibrosis in HCC and NAFLD Patients

( Se Young Jang ),( Won Young Tak ),( Soo Young Park ),( Young-oh Kweon ),( Yu Rim Lee ),( Bina Jeong ),( Sangkyung Seo ),( Gyoun-eun Kang ),( Gyeonghwa Kim ),( Keun Hur ),( Heon Tak Ha ),( Jae Min Ch 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: Mac-2 binding protein glycan isomer (M2BPGi) is recently identified as a useful non-invasive biomarker for the diagnosis of liver fibrosis. This study aimed to evaluate the diagnostic efficacy of serum M2BPGi for liver fibrosis in hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD) patients. Methods: M2BPGi levels were analyzed in serum samples collected from biopsy-proven HCC (n=135) and NAFLD (n=113) patients. Fibrosis was graded histopathologically in non-tumorous portion of HCC and NAFLD. Serum M2BPGi levels were determined with an automated immunoassay analyzer. Spearman’s correlation and Kruskal-Wallis test were used to evaluate the correlation and comparison among groups. Diagnostic efficacy for fibrosis was evaluated by the area under the receiver operating characteristic curve (AUC). Results: Median levels (range) of M2BPGi in HCC and NAFLD patients were 1.21 (0.12-14.33) cut-off index (COI) and 0.59 (0.13-5.90) COI, respectively. In HCC patients, fibrosis stages were 0 (n=22), 1 (n=10), 2 (n=11), 3 (n=16), and 4 (n=76). The M2BPGi levels showed a significant positive correlation (r= 0.436, P<0.001) with fibrosis grade in HCC patients and yielded the lower AUC value, 0.787 (P< 0.001) than transient elastography (TE), AUC value, 0.806 (P=0.030) to predict advanced fibrosis (F >2). In NAFLD patients, fibrosis stages were 0 (n=22), 1 (n=34), 2 (n=28), 3 (n=19), and 4 (n=10). The M2BPGi levels showed a significant positive correlation (r=0.578, P<0.001) with fibrosis grade in NAFLD patients and yielded the higher AUC value, 0.824(P< 0.001) than TE, AUC value, 0.637(P=0.035) to predict advanced fibrosis (F >2). Conclusions: Serum M2BPGi can be a useful non-invasive biomarker for predicting fibrosis in HCC and especially in NAFLD patients.

Clinical Impact of Exosomal microRNA as a Novel Biomarker of Liver Fibrosis

( Young Chang ),( Jae-a Han ),( Suk Min Kang ),( Soung Won Jeong ),( Tom Ryu ),( Han Seul Park ),( Jeong-ju Yoo ),( Sae Hwan Lee ),( Sang Gyune Kim ),( Young Seok Kim ),( Hong Soo Kim ),( So Young Jin 대한간학회 2020 춘·추계 학술대회 (KASL) Vol.2020 No.1

Aims: Many approaches have been suggested for the non-invasive diagnosis of liver fibrosis, including the use of serum biomarkers and ultrasound-based elastography, but none has yet replaced liver biopsy. MicroRNAs (miRNAs) have been suggested as potential diagnostic tools for liver diseases. We investigated alterations in the expression of serum exosomal miRNAs with the progression of liver fibrosis and evaluated their clinical applicability as biomarkers. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at a large-volume academic hospital in Korea. Exosomes were extracted from serum samples, and next-generation sequencing (NGS) of miRNAs was conducted in patients from different stages of liver fibrosis. Differential expression of miRNAs was quantified using targeted real-time quantitative polymerase chain reaction (RT-qPCR). A model was derived to discriminate advanced fibrosis based on miRNA levels using multivariate logistic regression. The performance of this model was evaluated and compared using area under the receiver operator characteristic (ROC) curve (AUC) and De- Long’s test. Results: NGS data revealed the relationship between exosomal miR-122 expression and liver fibrosis progression. The level of miR-122 decreased as the pathologic fibrosis grade progressed from stage 0 to 4. Patients with biopsy-proven advanced fibrosis had significantly lower levels of exosomal miR-122 (P<0.001) than those without advanced fibrosis. Exosomal miR-122 exhibited a fair performance in discriminating advanced fibrosis with an AUC of 0.77, which improved to 0.86 in combination with fibrosis-4 score (FIB-4) and transient elastography (TE). This value was higher than that reported for any other non-invasive modalities, including TE (AUC of 0.80) or FIB-4 (AUC of 0.57) alone. In a subgroup of patients with a non-viral etiology of liver disease, the performance of exosomal miR-122 as a biomarker improved, evident from the increase in the AUC value to 0.87. In this subpopulation, the combination model of miR- 122, FIB-4, and TE showed the best discrimination ability (AUC of 0.90), which was significantly higher than that of TE alone (AUC of 0.83; DeLong’s test P=0.046). Inhibition of miR-122 expression increased the proliferation of the human hepatic stellate cell line, LX-2, and upregulated the expression of collagen- 1A, a-smooth muscle actin, fibronectin, and transforming growth factor-ß. Conclusions: Exosomal miR-122 may serve as a novel biomarker for discriminating advanced liver fibrosis, and its accuracy may enhanced in combination with other non-invasive tests such as FIB-4 and TE.