Project supported by the National Natural Science Foundation of China, No. 39670339.
Published online Dec 15, 1998. doi: 10.3748/wjg.v4.i6.497
Revised: November 2, 1998
Accepted: December 5, 1998
Published online: December 15, 1998
AIM: To study the effect of lipid (triglyceride and very low-density lipoprotein, VLDL) on proliferation and activation of rat hepatic stellate cells (HSC).
METHODS: HSC were isolated and cultured from liver of Wistar rats by in situperfusion with pronase and collagenase and density gradient centrifugation with Nycodenz. HSC proliferation was examined with MTT colorimetric assay.
RESULT: Triglyceride of 12.5 mg/L had a promoting effect on proliferation of HSC (P < 0.05), 25, 50, 100 and 200 mg/L had no effects (P > 0.05), but 400 mg/L had an inhibiting effect (P < 0.01). VLDL of 6.25 and 12.5 mg/L had no effect on proliferation of HSC (P > 0.05), but increased concentration of VLDL could promote the HSC proliferation (P < 0.05).
CONCLUSION: Lipid had an effect on proliferation of HSC. Triglyceride and VLDL may promote HSC proliferation and may be associated with fatty liver and hepatic fibrogenesis.
- Citation: Lu LG, Zeng MD, Li JQ, Hua J, Fan JG, Fan ZP, Qiu DK. Effect of lipid on proliferation and activation of rat hepatic stellate cells (I). World J Gastroenterol 1998; 4(6): 497-499
- URL: https://www.wjgnet.com/1007-9327/full/v4/i6/497.htm
- DOI: https://dx.doi.org/10.3748/wjg.v4.i6.497
Despite the early controversy over the primary cellular source of extracellular matrix proteins in liver fibrosis, compelling in vitro and in vivo experimental evidence is now available to implicate activated hepatic stellate cells (HSC) in the pathogenetic role[1-3]. Mechanisms by which collagen-producing cells are activated under pathological conditions remain unknown and continue to be a topic of research interest[1,2,4,5]. Although the role of hepatocytes in lipid metabolism and transportation have been discussed intensively, that of HSC was not known More and more studies showed that HSC took part in lipid metabolism and transportation, their abnormality was related to the pathogenesis of fatty liver and liver fibrosis. In order to seek a possible explanation for the role of lipid in activation of HSC in liver fibrogenesis, we observed the effects of triglyceride and very low-density lipoprotein (VLDL) on proliferation of rat HSC.
Male Wistar rats, weighing 400 g-450 g were fed ad libitum with standard rodent chow.
HSCs were prepared by the methods of Friedman et al and Baroni et al with slight modifications. The rats were anesthetized with intraperitoneal pentobarbital (30 mg/kg). The liver was perfused in situ through the portal vein with 500 mL of calcium-free Gey’s balanced salt solution for 10 min at a flow rate of 40 mL/min-50 mL/min and then enzymatically digested with perfusate containing 0.05% collgenase (Sigma) and 0.1% pronase E (Merck) for 10 min-15 min. After being removed, the liver was cut into small pieces and incubated in 50 mL fresh GBSS-BSA containing 0.05% collagenase and 0.1% pronase E and stirred at 37 °C for 30 min. After passing through gauze, cell suspension was centrifuged at 500 × g for 7 min, the supernatant discarded at the cells washed twice further with Dulbeco’s modified Eagle’s medium (DMEM) (Gibco). The cell suspension was mixed with 18% (W/V) of Nycodenz (Sigma) in GBSS without NaCl. The gradient was centrifuged at 1450 × g for 17 min at 4 °C. The white, diffuse, flufly band in the lower region of the DMEM layer just above the Nycodenz cushion, which contained highly enriched HSC, was gently aspirated, diluted in about 30 mL DMEM and centrifuged at 450 × g for 7 min at 4 °C. The cell pellet was suspended in the incubation medium and seeded in the culture flasks with DMEM containing 20% fetal calf serum. Sterile condition was maintained during the entire isolation and purification procedures. Cell viability was assessed by trypan blue exclusion, and cell counting were conducted in a hemocytometer.
Purified HSC suspended in DMEM containing HEPES (15 mmol), penicillin (100 U/mL), streptomycin (100 mg/mL) and fetal calf serum (20% V/V) were seeded at a density of 1 × 105 cells/cm2 in the culture flasks. The medium was changed 20 to 24 h after plating, and every 3 to 4 d thereafter. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. To evaluate the purity of the cultures, HSC at day 2 or 3 and 7 after plating were tested by immunohistochemistry staining for desmin, lysozyme and factor VIII-related antigen, and by ultraviolet excited fluorescence microscopy at the length of 328 nm.
HSC proliferation was studied by colorimetric MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (Sigma) assay. HSC (1 × 105 cells/100 μL) were cultured in multiwell tissue culture plates (96 well/plate) (Corning, New York, NY, USA) for 2 d and 48 h in the presence or absence of triglyceride and VLDL. Twenty μL of MTT solution (5 mg/L) was added to all wells of an assay, and plates were incubated at 37 °C for 4 h. Then 100 μL dimethyl sulfoxide was added to all wells and mixed thoroughly to dissolve the dark blue crystals. After 20 min-30 min at room temperature to ensure that all crystals were dissolved, the plates were read on a E-Liza Mat-300 reader, using a test wavelength of 570 nm, a reference wavelength of 630 nm. Plates were normally read within 1hr of adding the dimethyl sulfoxide. All samples were analyzed in pentaplicate.
All data were expressed as mean ± SD. Statistical differences were assessed by the standard t test and P values of < 0.05 were judged to be statistically significant.
The yield of HSC ranged from 5 × 106 to 1 × 107 cells per liver. HSC displayed fading green-blue fluorescence with fluorescence microscopy at a wave length of 328 nm. After the first washing at 24 h, the HSC purity in culture exceeded by 95% as assessed by phase contrast microscopy and ultraviolet excited fluorescence microscopy. Viability of HSC assessed by trypan blue exclusion exceeded by 95%. HSC positive for desmin by immunohistochemistry in primary culture was above 90%, and in subculture above 95%. Lysozyme and factor VIII-related antigen were negative for HSC.
Trigylceride was directly administered to HSC at concentrations of 12.5, 25, 50, 100, 200 and 400 mg/L, respectively. The HSC proliferation was measured with colorimetric MTT assay. HSC proliferation in the presence of triglyceride is shown in Table 1. Compared with the contrast (0.1395 ± 0.0276), 12.5 mg/L of triglyceride had a promoting effect on proliferation of HSC (P < 0.05), 25, 50, 100 and 200 mg/L had no effects (P > 0.05), but 400 mg/L had an inhibiting effect (P < 0.01).
VLDL was directly administered to HSC at concentrations of 6.25, 12.5, 25, 50 and 100 mg/L of VLDL, respectively. The HSC proliferation was measured with colorimetric MTT assay. HSC proliferation is shown in Table 2. Compared with the contrast (0.1395 ± 0.0276), 6.25 and 12.5 mg/L of VLDL had no effect on proliferation of HSC (P > 0.05), but 50 and 100 mg/L of VLDL could promote HSC proliferation (P < 0.05 or P < 0.01).
There is now overwhelming evidence that HSC, which reside in the space of Disse, are the principal effectors of hepatic fibrogenesis. However, in contrast to most other conditions, it is far from clear whether the characteristic phenotypic transformation, proliferation and fibrogenesis by HSC is always a response to liver cell injury/or inflammation.
HSCs were demonstrated to present lipoprotein receptor in their surface, contained a large amount of fat droplets in their cytoplasm, and synthesized and secreted apolipoproteins. HSCs were in direct contact with plasma in the Disse’s space, accessable to both chylomicron and VLDL triglyceride, and do not need to transport the enzyme to the adjacent endothelium. All these suggested that HSC and hepatocytes were necessary for lipid mobilization and transportation[1,2]. Their abnormality was related to the disturbance of lipid mobilization and transportation which made triglyceride concentrate in the liver, inducing fatty liver and liver fibrosis. Reeves et al’s study showed that the mechanism of HSC activation/proliferation and subsequent fibrogenesis related to alcohol may be unrelated to necroinflammation, and that HSC activation occurced in the absence of hepatitis in alcoholic liver disease and correlated with the severity of steatosis. However, isolated fatty liver without hepatitis occurs more frequently than does steatohepatitis. Thus it is not clear whether the accumulation of fat in the liver is responsible for inflammation or whether inflammation evoked by some stimulus caused cell dysfunction that resulted in steatosis. Vicente et al studied the lipid metabolism during in vitro induction of the lipocyte phenotype in HSC. HSC can produce and bind lipoprotein lipase to their own surface, using it potentially for processing exogenous lipids in the acute phase of lipocyte induction, when the accumulation of lipids was accelerated. In the our study, we found that triglyceride and VLDL promoted HSC proliferation at certain concentrations. Some animal studies also confirmed that steatosis and collagen content in experimental liver cirrhosis are affected by dietary monounsaturared and polyunsaturated fatty acids[12,13]. The great amount of lipid in diet produced fatty liver and liver fibrosis more easily. These experimental evidence indicated that the infiltration of lipid increased the production of fibrous tissue in the liver. The earliest event in the development of fibrosis appears to be activation of HSC by such factors as lipid peroxides[14,15]. This caused proliferation of HSC and initiation of fibrogenic cascade in the liver. In addition, the investigation showed that high concentration of triglyceride (400 mg/L) had an inhibiting effect on HSC proliferation. It was deduced that high concentration of triglyceride might have a toxic effect on HSC. The precise role of triglyceride and VLDL in the HSC proliferation and activation needs to be addressed in futher studies.
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