Changes of portal hemodynamics in the development of experimental liver cirrhosis

AIM: To understand the formation of portal hypertension through the change of portal hemodynamics on experiment cirrhosis. METHODS: Carbon tetrachloride was subcutaneously injected in the rat. The changes of the portal hemodynamics in the pathological process of liver tissue were observed. RESULTS: The liver underwent degeneration, necrosis of hepatocytes, and the normal architecture of the liver lobules was replaced by pseudolobule, which consist of regenerative hepatocytes and fibrous septa. The diameter, the blood flow velocity and the blood flow quantity of portal were significantly higher than that in former group (P＜0.05 or P＜0.01) two weeks after the injection of carbon tetrachloride. In the fifteenth week, these parameters were lower than that before owing to the forming of portacaval collateral circulation (P＜0.01). The congest index of the portal in second week, fifth week and fifteenth week were statistically higher than its predecessor (P＜0.05 or P＜0.01), except that in tenth week, which had no statistical significance (P>0.05). CONCLUSION: The changes in hemodynamics of the portal are in accordance with the changes in pathology of liver in the formation of liver cirrhosis.     

Changes of liver sinusoidal endothelial cells and basement membrane in early stage of liver fibrosis induced by carbon tetrachloride in rats

AIM：To explore the development of hepatic sinusoidal capillarization in the early stage of liver fibrosis induced by carbon tetrachloride (CCl4) in rats. METHODS：Clean SD rats were randomly divided into normal control group (group N, n=6) and liver fibrotic model group (group M, n=32). The rats in group N were intraperitoneal injected with saline and the rats in group M were intraperitoneal injected with CCl4 (2 mL/kg, twice a week for 4 weeks). At the end of the 3rd day and the 1st, 2nd and 4th weeks, all rats were killed and then the samples were collected. The pathological changes in the livers were observed by HE staining and Masson straining. The development of hepatic sinusoidal capillarization was observed by transmission electron microscopy (TEM) and immunohistochemical staining. The cell surface expression of vascular endothelium-associated marker CD31, collagen type Ⅳ (Col IV) and laminin (LN) was determined. RESULTS：HE and Masson staining showed the formation of liver fibrosis after treatment with CCl4 for 4 weeks. TEM showed that the fenestrate diameter of liver sinusoidal endothelial cells (LSECs) grew down, the fenestrate numbers of LSECs were decreased along with the development of liver fibrosis, and the consecutive basement membrane was formed at the end of the experiment. The expression of CD31 was significantly increased along with the development of defenestration, and the expression of Col IV and LN was significantly increased after the treatment with CCl4 for 2 weeks and 4 weeks, respectively. CONCLUSION：The typical hepatic sinusoidal capillarization was detected in the early stage of liver fibrosis, and the deposition of LN in the liver sinusoidal walls was the mainly factor of formation of the consecutive basement membrane.     

Effect of pulmonary vascular endothelial cells on the development of acute lung injury in rats

AIM: Effect of endothelial cell on the development of acute lung injury and the prevention of dexamethasone in acute lung injury were observed.METHODS:Rats were divided into three groups:1.Control group.2.LPS group:Venous injection with LPS(5mg/kg body weight),execute respectively at 1 h,2 h,6 h and 24 h after LPS injection. 3.dexamethasone group:intraperitoneal injection with dexamethasone ,1 h before LPS injection,execute after 2 hours after LPS injection.RESULTS: Serum NO,TNF-α levels,lung iNOS activity and lung ICAM-1mRNA expression were increased( P <0.05, P <0.01, vs control group),but serum ACE was decreased( P <0.01).Dexamethasone could improve all the changes above mentioned.CONCLUSION:Endothelial cell played a vital role in the development of acute lung injury and dexamethasone could prevent acute lung injury.     

Role of 78 kD glucose-regulated protein in the development of liver cirrhosis promoted by intestinal endotoxemia in rats

AIM: To explore the role of 78 kD glucose-regulated protein (GRP78) in the development of liver cirrhosis in rats promoted by intestinal endotoxemia (IETM). METHODS: Fifty-one male Wistar rats were randomly divided into liver cirrhosis groups of 4th-week, 6th-week and 8th-week, and normal control group at the corresponding time points. The rat model of hepatic cirrhosis was induced by employing multiple pathogenic factors to the animals. The liver injury and hepatic fibrosis were observed with the staining of HE and VG, respectively. The expression of GRP78 at the mRNA and protein levels was measured by the methods of RT-PCR and immnunohistochemistry, respectively. The concentrations of alanine aminotransferase(ALT), endotoxin, TNF-α and homocystine (HCY) in plasma, and the content of TNF-α, malondialdehyde(MDA) and PⅢP in liver tissues were detected. RESULTS: As liver cirrhosis developed, the levels of ALT, endotoxin, TNF-α and HCY in plasma, the expression of GRP78 at mRNA and protein, the content of TNF-α, MDA and PⅢP in liver tissues, and the index of liver fibrosis were gradually increased and were significantly higher than those in normal control group (P<0.05). Elevated endotoxin in plasma was correlated positively with the protein expression of GRP78, the content of MDA and HCY in plasma and the index of liver fibrosis (P<0.01). Elevated protein expression of GRP78 was correlated positively with the content of MDA and HCY in plasma and the index of liver fibrosis (P<0.01). CONCLUSION: GRP78 plays an important role in the development of liver cirrhosis. Endoplasmic reticulum stress is a possible mechanism in the development of liver cirrhosis promoted by IETM.     

Effects of macrophage polarization during development of liver cirrhosis in rats

AIM: To explore the state of macrophage polarization and its relation with intestinal endotoxemia-endoplasmic reticulum stress in the development of liver cirrhosis induced by multiple pathogenic factors in rats. METHODS: The male SD rats (n=36) were randomly divided into normal control group and liver cirrhosis model group, and sacrificed at the end of the 4th, 6th and 8th weeks. The rat model of liver cirrhosis was induced by multiple pathogenic factors. The levels of alanine aminotransferase (ALT), endotoxin, homocysteine (Hcy) in the plasma, and inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), arginase-1 (Arg-1) and interleukin-10 (IL-10) in the liver tissues were detected by ELISA. Histopathological change of the liver was observed under microscope with the staining of hematoxylin and eosin (HE) and van Gieson (VG). The expression of glucose-regulated protein 78 (Grp78), nuclear factor-kappa B (NF-κB), interferon-regulatory factor 5 (IRF5), CD86, CD206 and transforming growth factor-β1 (TGF-β1) at mRNA levels in the liver tissues were detected by the method of real-time fluorescence quantitative PCR.RESULTS: Compared with the corresponding normal control group, the levels of ALT, endotoxin, Hcy in the plasma and Grp78 mRNA in the liver tissues in liver cirrhosis model group were significantly and gradually increased (P<0.05). The mRNA expression of NF-κB, IRF5 and CD86, and the protein levels of iNOS, TNF-α and IL-6 in the liver tissues were significantly increased (P<0.05), and they successively increased from the 4th week to the 6th week and decreased reversely at the 8th week. The mRNA expression of CD206, TGF-β1, Arg-1 and IL-10 in the liver tissues were significantly increased from the 6th week to the 8th week (P<0.05), and no significant difference at the 4th week was observed. The level of endotoxin in the plasma was correlated with the mRNA expression of Grp78 in the liver tissues (P<0.01). Both endotoxin in the plasma and Grp78 mRNA in the liver tissues were correlated with the mRNA expression of CD86 and CD206 in the liver tissues (P<0.01).CONCLUSION: The pathway of liver damage-intestinal endotoxemia-endoplasmic reticulum stress-macrophage polarization may be critical in the pathogenesis of liver cirrhosis induced by multiple pathogenic factors.     

Role of endothelin in portal hypertension induced by endotoxin

AIM:To study the role of endothelin-1 (ET-1) in portal hypertension (PHT) induced by endotoxin. METHODS:Collagenase in situ perfusion was adopted to separate hepatic stellate cells (HSCs). HSCs was cultured on concretized collagen. ET-1 anti-sense oligonucleotide was added into the culture medium and then LPS was also added up to the concentration of 1 000 μg/L. The diameters of the concretized collagen were measured. Sense and mis-sense oligonucleotide were applied as control. ET-1 in the culture medium was detected by radioimmunoassay and ET-1 mRNA in HSCs was detected by RT-PCR. β-actin of HSCs was detected by Western blotting. RESULTS:The diameter of concretized collagen on which HSCs pretreated with ET-1 anti-sense oligonucleotide was 93.3%±3.8% the size of the primary. The diameter of concretized collagen of the control groups were 70.1%±4.8% and 70.5%±3.9% (P<0.05). ET-1 was (49.8±7.4)ng/L in the culture medium of HSCs pretreated with ET-1 anti-sense oligonucleotide and (329.8±34.9), (339.1±43.7)ng/L in the control medium (P<0.05). β-actin and ETI mRNA presented in HSCs pretreated with ET-1 anti-sense oligonucleotide was much less than that in the controls. CONCLUSION:ET-1 anti-sense oligonucleotide inactivated HSCs by counteracting the expression of ET-1, which may be helpful to control PHT induced by LPS.     

Role of nitric oxide in the development of glomerular ischemia reperfusion injury in rats

AIM:To investigate the effects of nitric oxide on ultrastructure and anionic sites of glomerular in renal ischemia reperfusion injured (I-RI) rats. METHODS:Animals were divided randomly into five groups:(1) sham group (n=6);(2) I-RI group (n=6), 0.3 mL normal saline was injected via venae lingualis 20 min before ischemia;(3) SNP+I-RI group (n=6), 2.5 μg/kg sodium nitroprusside (SNP) was injected via venae lingualis 20 min before ischemia;(4) AG+I-RI group (n=6), 10 mg/kg aminoguanidine (AG) was injected via venae lingualis 20 min before ischemia;(5) L-NNA+I-RI group (n=6), 10 mg/kg Nω-nitro-L-arginine (L-NNA) was injected via venae lingualis 20 min before ischemia. Anionic sites of glomerular were studied with a cationic probe-polyethyleneimine (PEI) and ultrastructure was observed under electron microscope in renal I-RI rats. RESULTS:(1) Ultrastructure of glomerular was normal and anionic sites (AS) was located clearly in lamina rare externa of GBM in sham rats. The PEI particles arranged regularly in line (19.3±1.7/1 000 nm) under electronic microscope. Obvious foot processes derangement and effacement were observed and the AS number in GBM of I-RI group was fewer (16.6±1.0/1 000 nm, P<0.05) and the particle was smaller than that in sham group. (2) Compared with I-RI group, the foot process effacement was aggravated in SNP+I-RI group and L-NNA+I-RI group. SNP caused the numbers of anionic sites reduced after renal I-RI (11.7±3.2/1 000 nm, P<0.05), and the electronic density of the PEI granule was also reduced. AG lead a increase in anionic site number (17.8±1.0/1 000 nm, P<0.05), but still fewer than that in sham group (P<0.05). The numbers of anionic sites was not changed in L-NNA+I-RI group (14.7±0.9/1 000 nm, P>0.05). CONCLUSION:Foot process effacement and reduction of anionic sites were present in glomerular filtration membrane in renal I-RI rats. NO aggravated those injuries, indicating that NO plays a role in the ultrastructure damages of glomerular filtration membrane in I-RI rats.     

Role of autophagy in ursolic acid-induced injury of human umbilical vein endothelial cells

AIM: To investigate the role of autophagy in the injury of human umbilical vein endothelial cells (HUVECs) induced by ursolic acid (UA). METHODS: HUVECs were cultured in vitro with UA at various concentrations for 36 h and the proliferation inhibitory rate of HUVECs was determined by MTT method. The change of ultrastructure was observed under transmission electronic microscope (TEM). The autophagy was observed using fluorescent microscope by monodansylcadaverin (MDC) staining. The protein level and mRNA expression of microtubule-associated protein light chain 3(LC3) and Beclin-1 were detected by Western blotting and RT-PCR, respectively. Cell apoptotic rate was measured by flow cytometry analysis. RESULTS: UA at various concentrations showed significantly dose-dependent inhibitory effect on the proliferation of HUVECs. Autophagy was induced in HUVECs treated with UA as detected by MDC staining and TEM. The protein level and mRNA expression of LC3 and Beclin-1 in HUVECs were significantly increased following the treatment with UA, which was also in a time-dependent manner. Compared with UA group, addition of 3-methyladenine(3-MA) inhibited the increase in autophagic vacuoles and exacerbated the apoptosis. CONCLUSION: Autophagy shows protective effect on the proliferation inhibition of HUVECs induced by UA and the proliferation inhibition can be enhanced by the autophagy inhibitor 3-MA. 3-MA may enhance the apoptotic rate of HUVECs induced by UA.     

Role of caspase-3 dependent hepatocyte apoptosis in liver ischemia-reperfusion injury in cirrhotic rats

AIM:To investigate whether hepatocyte apoptosis is contributed to liver ischemia-reperfusion (I/R) injury and the relationship between liver caspase-3 activity and hepatocyte apoptosis in cirrhotic rats. METHODS:Liver ischemia-reperfusion is induced by Pringle maneuver. The cirrhotic rats were randomized into two groups: Group A: simple hepatic blood inflow occlusion (HBIO); Group B: HBIO + inhibitor, before HBIO, ZVAD-fmk 15 mg/kg was injected via dorsal penis vein; Group C: healthy rat, simple HBIO. The ischemia time was 30 min in these groups. Serum aspartate aminotransferase(AST), liver caspase-3 activity, and apoptotic hepatocytes were examined in the three groups. RESULTS: After 6 h of reperfusion, the liver caspase-3 activity was markedly elevated and reached its peak, which was statistically higher than that of before I/R . The same change occurred in hepatocyte apoptosis between 6 h of reperfusion and before I/R (20.9%±4.9% vs 0.5%±0.3%, P＜0.01). As the reperfusion prolonged, the caspase-3 activity and apoptotic hepatocyte decreased gradually. The 7th-day survival rate was 62.5% in group A. The serum AST, liver caspase-3 activity and apoptotic hepatocytes were significantly higher in group A than those in group B and C, representing the most severe liver injury among the three groups. CONCLUSION:Hepatocyte apoptosis is the major form of cell death in liver ischemia-reperfusion injury in cirrhotic rats. Hepatoctye apoptosis induced by I/R is caspase-3 dependent, and inhibiting caspase-3 can alleviate liver injury. The caspase-3 dependent hepatocyte apoptosis is highly contributed to the pathological phenomenon that the ischemic sensitivity of cirrhotic liver is higher than normal liver.     

Role of hydrogen sulfide in alleviation of liver injury by mesenteric lymph drainage in hemorrhagic shock rats

AIM：To investigate the role of hydrogen sulfide (H2S) in alleviation of liver injury by mesenteric lymph drainage in hemorrhagic shock rats. METHODS：A hemorrhagic shock model was established in male Wistar rats. DL-propargylglycine (PPG), an inhibitor of cystathionine γ-lyase (CSE) which is a synthase of H2S, or sodium hydrosulfide (NaHS), a donor of H2S, was administered to the hemorrhagic shock rats with mesenteric lymph drainage. The rats were randomly divided into sham, shock, shock+drainage, shock+drainage+PPG (45 mg/kg, ip, 0.5 h before hemorrhage) and shock+drainage+NaHS (28 μmol/kg, ip, 0.5 h before hemorrhage) groups. Fluid resuscitation was performed 1 h after hypotension, and then mesenteric lymph was drained in the rats of shock+drainage, shock+drainage+PPG and shock+drainage+NaHS groups for 3 h. The hepatic histomorphology was observed. The biochemical indexes of hepatic function in plasma, and H2S, CSE, Toll-like receptor 4 (TLR4), interleukin (IL)-10, IL-12 and tumor necrosis factor α (TNF-α) in hepatic homogenate were also examined. RESULTS：The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bile acid (TBA) in plasma, and H2S, CSE, TLR4, IL-10, IL-12 and TNF-α in hepatic homogenate in shock group were significantly higher than those in sham group. Mesenteric lymph drainage obviously reduced these indexes in shock rats, except for TLR4. PPG further decreased these indexes except for CSE, while NaHS increased these indexes except for TBA and CSE. Morphological observation showed that liver injury appeared in the rats from shock and shock+drainage+NaHS groups, and there was nearly normal hepatic structure in the rats from sham, shock+drainage and shock+drainage+PPG groups. CONCLUSION：The mechanism of mesenteric lymph drainage alleviating liver injury in hemorrhagic shock rats is related to reducing the production of H2S and alleviating the H2S-mediated inflammation.     

Role of endothelial cell glucose metabolism in vascular development

As an important organ in the body, blood vessels transport oxygen and nutrients to the tissues of the body. Endothelial cells are layers of cells lined with blood vessels whose metabolism is involved in regulating multiple links of vascular development and vascular diseases. Glucose metabolism provides the main energy for endothelial cells, and changes in glucose metabolism regulation affect vascular development and even lead to various diseases. Recently, strategies have been proposed to target endothelial cell metabolism for the treatment of vascular diseases. This article reviews the role of endothelial cell glucose metabolism in vascular development and vascular diseases in recent years.     

Effect of portal vein transplantation with allogeneic adipose tissue-derived mesenchymal stem cells on liver fibrosis in SD rats

AIM: To explore the influence of adipose tissue-derived mesenchymal stem cells (ADMSCs) transplantation on hepatic fibrosis in rats. METHODS: ADMSCs from abdominal lipid tissues were extracted, cultured and passaged. The hepatic fibrosis rat model was built up and randomly divided into 3 groups: hepatic cirrhosis group (n=14); portal vein transplantation group (n=11) and caudal vein transplantation group (n=14). Computer tomography(CT) perfusion index, histological scores and microvessel density were detected and compared after transplantation of ADMSCs among the 3 groups. RESULTS: After transplantation of ADMSCs, the total hepatic blood perfusion, especially portal vein perfusion, significantly increased in portal vein transplantation group determined by CT perfusion scan (P<0.05), but slightly increased in caudal vein transplantation group. The histological scores showed significant alleviation of fibrosis evidence in portal vein transplantation group, and slightly change of adipose degeneration in caudal vein transplantation group. Microvessel density decreased significantly in portal vein transplantation group as compared to the other 2 groups (P<0.05). CONCLUSION: Transplantation of ADMSCs greatly helps the alleviation of hepatic fibrosis. Portal vein transplantation benefits more than caudal vein transplantation.     

The dynamic changes of plasma nitric oxide and endothelin-1 in prehepatic portal hypertension rats

AIM:To observe the dynamic changes of plasma levels of nitric oxide(NO) and endothelin (ET-1) in portal veins of the rats during prehepatic portal hypertension, and investigate the role of them in hyperdynamic circulation.METHODS:The models of prehepatic portal hypertension were established in Sprague-Dawley rats by means of partial portal vein ligation (PVL). The plasma levels of nitrite/nitrate (NO₂^-/NO₃^-) and ET-1 in the portal veins were detected by the method of nitric reductase and radioimmunoassay, respectively. In this study, rats were divided into normal, sham operation (SO) and PVL group. SO and PVL rats were divided into several subgroups according to different time after operations. Meanwhile, the changes of several hemodynamic indexes in these rats were also measured.RESULTS:The levels of NO₂^-/NO₃^- were significantly increased and ET-1 were significantly decreased in rats at different time after PVL compared with normal control, whereas the hemodynamic indexes changed accordingly.CONCLUSION:The portal hypertensive rats are in hyperdynamic circulatory state (HCS). NO and ET-1 may play an important role in the induction and maintenance of HCS.     

Protective effect of famotidine against gastric mucosa damage resulting from interdicted hepatic portal in the liver cirrhosis rat

AIM: To investigate the influence of hepatic portal interdicting on gastric mucosa of the liver cirrhosis rats and the protective effect of famotidine on gastric mucosa. METHODS: Fifty Wistar rats were randomly divided into 5 groups; group A (healthy rats), group B (healthy rats with interdicted hepatic portal), group C (liver cirrhosis rats without interdicted hepatic portal), group D (liver cirrhosis rats with interdicted hepatic portal) and group E (liver cirrhosis rats without interdicted hepatic portal and use of famotidine); The gastricfluid pH, gastric mucous content (GMC), gastric mucosa blood flow (GMBF) and the damage index of gastric mucosa (DIGM) of the every group were observed. RESULTS: There is no significant difference in the above parameters between group A and group B (P>0.05). Compared with group A, GMBF of group C, D, and E were reduced (P<0.01), pH, GMC of group C, D and E were also reduced (P<0.05); DIGM of group C, D and E were increased (P<0.01). All the changes of group D were more obvious than those of group C (P<0.01); While there was no significant difference in all the parameters between group E and group C (P>0.05). CONCLUSION: Hepatic portal interdicting can aggravate the gastric mucosa damage of the liver cirrhosis rat, and famotidine can protect against such gastric mucosa injury through improving the microcirculation of gastric mucosa.     

Role of endogenous nitric oxide in apoptosis of alveolar epithelial cells in the development of pulmonary fibrosis in rats

AIM: To study the role of high level of endogenous nitric oxide (NO) in apoptosis of alveolar epithelial cells in the development of pulmonary fibrosis in rats. METHODS: The content of nitrite/nitrate (NO₂^-/NO₃^-) in out-flowing pulmonary blood (OPB) was assayed by nitric acid reduction method. The apoptosis of alveolar epithelial cells was observed by TdT-mediated dUTP nick-end labeling (TUNEL) and electron microscopy, respectively. The above indices were observed on the day 14 and the day 30 after intratracheal administration of BLMA₅ alone or along with blockade of iNOS by aminoguanidine (AG) in rats. RESULTS: (1) Both the content of NO₂^-/NO₃^- in OPB and the number of apoptotic alveolar epithelial cells in lung were increased in BLMA₅ 14 d group, compared with normal control group and BLMA₅ 30 d group, respectively (P<0.05). The high level of NO₂^-/NO₃^- in OPB and the apoptosis of alveolar epithelial cells were ameliorated by AG. CONCLUSION: The apoptosis of alveolar epithelial cell is induced by high level of endogenous NO in the development of pulmonary fibrosis.     

Role of MCU in bupivacaine-induced spinal cord neurotoxic injury in diabetic rats

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in bupivacaine (B)-induced spinal cord injury in diabetic (D) rats.METHODS: Healthy male Sprague-Dawley rats, weighing 180~220 g, were divided into normal rats and diabetic rats. After intraperitoneal injection of streptozotocin for building diabetic rat mo-del, 48 male rats were randomly divided into 6 groups with 8 rats in each group as following:control (C) group (normal rats by intrathecal injection of normal saline), D group (diabetic rats by intrathecal injection of normal saline), C+B group (normal rats by intrathecal injection of bupivacaine), D+B group (diabetic rats by intrathecal injection of bupivacaine), D+R₁+B group (diabetic rats by intrathecal injection of 10 μmol/L Ru360 and bupivacaine) and D+R₂+B group (diabetic rats by intrathecal injection of 50 μmol/L Ru360 and bupivacaine). The changes of paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured before modeling, after modeling, and 12 h, 24 h and 48 h after intrathecal injection. Lumbar enlargement was removed from spinal cord after rats were killed. MCU expression was tested by RT-qPCR and Western bolt. The levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) were determined by ELISA. Spinal neuronal apoptosis was measured using TUNEL assay.RESULTS: Compared with D group, the expression of MCU, the values of PWMT and PWTL, the levels of MDA and 8-OHdG, and the apoptotic rate of spinal cord neurons were significantly increased in D+B group (P<0.05). Compared with D+B group, the expression of MCU, the values of PWMT and PWTL, the levels of MDA and 8-OHdG, and the apoptotic rate of spinal cord neurons were significantly decreased in D+R₂+B group (P<0.05).CONCLUSION: Bupivacaine enhances oxidative stress and aggravates spinal cord injury via up-regulating MCU activity in diabetic rats.