The 20-Year Natural History Of Chronic HCV Infection By Plasmapheresis And Its Sphingolipidomics Study

Background and Objectives:Chronic hepatitis C (CHC) may have different disease progression due to the different infection modes. As a unique mode of infection, the disease progression of CHC infected by plasma donation through plasmapheresis is rarely reported, even now there are scarcely any histologically-confirmed studies using liver biopsy to evaluate the fibrosis progression of about 20-year hepatitis C virus (HCV) infection. During the progression of liver disease, sphingolipids may be involved in the apoptosis of hepatic cells and onset of hepatic inflammation, affect the development of hepatic steatosis, or may contribute to the natural progression of liver disease. Currently rare studies report the role of sphingolipids in disease progression caused by chronic HCV infection. Therefore, the primary purpose of this study was, based on the methods of liver biopsy and sphingolipidomics, to clarify the progression of liver fibrosis in CHC caused by plasma donation through plasmapheresis after approximately 20-year HCV infection and identify factors which may affect the disease progression; to reveal the alterations of plasma sphingolipids and identify sphingolipids which have close relationship with hepatic inflammation, fibrosis, steatosis in disease progression of CHC; further to identify potential biomarkers for different disease statuses in the plasma sphingolipids; to clarify the role of sphingolipids’metabolism in the pathological mechanisms of hepatocytes’apoptosis initially.Methods:All recruited participants in the present study were from a cohort of 122 Han Chinese CHC without antiviral therapy. All the patients infected HCV by plasma donation through regular transfusion blood cell after plasmapheresis between 1992 to 1995. Patients underwent liver biopsies under ultrasound guidance. Metavir scoring system was performed to evaluate hepatic fibrosis stage. Scheuer scoring system was performed to evaluate hepatic inflammation grade. Hepatic steatosis were graded according to the proportion of intrahepatic lipids. The clinical routine data were collected. On basis of sphingolipidomics testing platform using high-performance liquid chromatography-mass spectrometry/mass spectrometry, plasma sphingolipids were detected in all patients who underwent liver biopsy and the relationship between plasma sphingolipids with liver inflammation, fibrosis, steatosis was explored. In basic research in vitro, siRN A technology was employed to block the gene expression of glucosykeramide synthase. MTT, AnnexinV-FITC/PI immunofluorescence, enzyme-linked immunosorbent assay, real-time quantitative PCR, Western blot methods were performed to detect hepatic cell proliferation and apoptosis after abnormal metabo lism o f glycosp hingo lip ids.Results:● A total of 120 patients had valid liver biopsies. One patient did not undergo liver biopsy due to cirrhosis related-ascites. The morbidity of cirrhosis was 2.5% in this cohort Hepatic inflammation was an independent factor for significant hepatic fibrosis (F ≥ 2) (odds ratio[OR]=5.02; 95% confidence interval[CI]2.17-11.63).● By high-performance liquid chromatography-mass spectrometry/mass spectrometry analysis, a total of 44 kinds of plasma sphingolipids were identified and quantified. For hepatic inflammation, in total CHC patients or patients with normal alanine aminotransferase (ALT) levels, hexosylceramide (HexCer) (d18: 1/22:0) was an independent factor of hepatic necroinflammation, and its adjusted OR were 1.01 (95% CI:1.0-1.02) and 1.02 (95% CI:1.01-1.03), respectively. As for the ability of distinguishing hepatic necroinflammation in CHC patients with normal ALT, among the sphingolipids and conventional indicators, HexCer (d18:1/22:0) had the largest area under the curve (AUC) value.● For hepatic fibrosis, in CHC population and CHC with significant fibrosis (F ≥ 2) populations, HexCer (dl8:1/12:0) was closely related with severe hepatic fibrosis (F ≥ 3), and had a certain ability to distinguish severe liver fibrosis, especially that the ability showed an upward tendency after the combination with conventional indicators.● For hepatic steatosis, plasma sphingomyelin (SM) (d18:1/22:0) was an independent and significant factor of hepatic steatosis, and the adjusted OR was 1.12 (95% CI:1.01-1.24), in patients with HCV gene type 2, SM (d18:1/22:0) was also independently associated with hepatic steatosis (OR = 1.21; 95% CI: 1.04-1.40), its AUC for distinguishing hepatic steatosis in gene type 2 CHC was 0.726 (95% CI:0.581-0.872).● The in vitro basic experiments showed that after interference of glucosylceramide synthase gene expression, the proliferation of hepatic cells was significantly inhibited, with obvious apoptosis phenomenon, and the Bc1-2; mRNA expression decreased, Bax mRNA expression levels increased, expression of Caspase 3 mRNA and protein upregulated.Conclusions:● According to the results of liver biopsies, the morbidity of cirrhosis of HCV infection by plasma donation through plasmapheresis for approximately 20 years is low. Hepatic inflammation is closely related to the occurrence of significant liver fibrosis. The decline of liver inflammation may delay progression of liver fibrosis.● In the natural progression of CHC, the plasma sphingolipid HexCer(d18:1/22:0) has a close relation to hepatic necroinflammation and shows preferable potential for the diagnosis of hepatic necroinflammation, especially for CHC patients with normal ALT. Plasma HexCer (d18:1/12:0) might be related to severe liver fibrosis in the CHC population, particularly in patients with significant liver fibrosis. SM (d18:1/22:0) may be a novel biomarker for liver steatosis in HCV gene type 2 CHC patients.● Basic research suggests that metabolic changes of sphingolipids caused by the lack of glucosylceramide synthase gene expression may be involved in the proliferation and apoptosis of liver cells.