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Evidence-Based Complementary and Alternative Medicine
Volume 2012 (2012), Article ID 786365, 8 pages
Salvianolic Acid B Reducing Portal Hypertension Depends on Macrophages in Isolated Portal Perfused Rat Livers with Chronic Hepatitis
1Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China
2School of Basic Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
Received 9 July 2012; Accepted 29 August 2012
Academic Editor: Youn Chul Kim
Copyright © 2012 Xin Zhao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This study is aimed to investigate the effects of Sal B on portal hypertension (PH). PH with chronic hepatitis was induced by carbon tetrachloride (CCl4) in rats. The model was confirmed with elevated portal pressures and increased serum CD163 levels. The inducible nitric oxide synthase (iNOS) or heme oxygenase-1 (HO-1) in portal triads was assessed. The isolated portal perfused rat liver (IPPRL) was performed at , , , and in the progression of chronic hepatitis. After constricting with phenylephrine, the portal veins were relaxed with Sal B. The EC50 of Sal B for relaxing portal veins was , , , and mol/L at d0, d28, d56, and d84, respectively. More macrophages infiltrated in portal triads and expressed more iNOS or HO-1 as PH advanced. The areas under the curve (AUCs) of Sal B for reducing PH were positively correlated with the levels of iNOS or HO-1 in portal triads, and so did with serum CD163 levels. Sal B reduces PH in IPPRL with chronic hepatitis, via promoting portal relaxation due to macrophage-originated NO or CO in portal triads, partly at least.
Portal hypertension (PH) is a common complication in the patients with advanced chronic hepatitis . The increased hepatic vascular resistance and portal hyperemia are involved in the reversible pathogenesis as the potent therapy targets .
Salvianolic acid B (Sal B) is a molecule from the root of Salvia miltiorrhiza (Danshen), which is a traditional Chinese medicine widely used for cardiovascular diseases . Sal B is effective for liver fibrosis and PH in patients  or animals . In the -induced PH rats, Sal B could inhibit the constriction of hepatic stellate cells . However, our previous study indicated that Sal B constrict portal veins of the isolated portal perfused rat livers (IPPRLs) at physiological status . The underlying mechanisms of Sal B for PH remain unclear.
It is reported that nitric oxide (NO) and carbon monoxide (CO) play key roles in the pathogenesis of PH . Both signal molecules directly relax portal veins through upregulation of cGMP via guanylate cyclase . NO from endothelial NO synthase (eNOS) aggravates PH through systemic hyperemia , and inducible NO synthase (iNOS) exacerbates PH by producing peroxynitrite (ONOO−) . It has been reported that the reduced NO bioavailability is involved in the increased hepatic vascular resistance . There is an increase of superoxide release by NADPH oxidase in liver with chronic hepatitis  and an overproduction of iNOS from macrophages . The iNOS-derived NO reacts with superoxide, leading to ONOO− formation, with a decrease in NO bioavailability . Heme oxygenase-1(HO-1) is a rate-limiting enzyme catalyzing heme to CO, iron, and biliverdin. Biliverdin is then converted to bilirubin, which acts as a highly effective antioxidant and free radical scavenger against oxidation . HO-1 also showed hepatoprotection against ischemia-reperfusion injury, endotoxemia, hyperoxia-induced hepatic injury, and immune-mediated apoptotic liver damage . Furthermore, HO-1/CO activation downregulates the inflammatory response by blocking the formation of ONOO− from iNOS . While the ONOO− induces HO-1 protein expression but mediating its inactivation .
Sal B has an effect on  production of NO or CO from activated macrophages  under inflammatory cytokines . In addition, Sal B could protect endothelia from the oxidation by blocking PI3K/Akt signal pathway . Therefore, Sal B was proposed to rescue NO bioavailability or to maintain CO potency from the macrophage at portal triads in advanced chronic hepatitis.
The purpose of present study is to investigate the effects of Sal B on PH in IPPRL with chronic hepatitis and to analyze further the NO or/and CO signals through the relationship between the Sal B potency and the existed iNOS or HO-1 from the macrophages in portal triads.
2. Materials and Methods
Carbon tetrachloride (CCl4), olive oil, and heparin sodium were purchased from Sinopharm Chemical Reagent Company. Acetylcholine chloride and phenylephrine hydrochloride were obtained from Sigma (USA). Salvianolic acid B (purity >99%) was purchased from Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
Thirty two six-week-old male Wistar rats (180–200 g) were purchased from Animal Centre of the Chinese Academy of Medical Sciences. All rats were kept under a 12 h/12 h light/dark cycle, temperature (°C), and humidity (%) controlled SPF environment. The rats were fed standard rodent pellets and allowed free access to filtered water. All experiment procedures were performed in accordance with the Guidelines of Animal Experiments from the Committee of Medical Ethics, National Health Department of China.
2.3. Induction of Portal Hypertension with Chronic Hepatitis
PH with chronic hepatitis was induced by CCl4 in rat as described previously (Figure 1) . Rats were injected subcutaneously with a mixture of 40% (v/v) CCl4 in olive oil (3 mL/kg) two times a week for 0, 28, 56, and 84 days, respectively [20, 21]; olive oil was the vehicle for age-matched control. Eighty four hours after the last CCl4 injection, rats were anesthetized with a subcutaneous injection of sodium pentobarbital (50 mg/kg). A midline incision was made to open abdominal cavity, and ascitic samples were collected and quantified as described preciously . The exuded liquid ratios were calculated as exuded liquid weight/body weight ×100. The portal pressure in vivo was recorded. The blood sample was collected for analyzing the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and albumin (Alb) levels by biochemistry and CD163 levels by immunoassay. Then the hepatic artery, portal vein, and hepatic vein were canalized [20, 21]. The liver, spleen, and kidneys were harvested, and the organ indexes were calculated as organ weight/body weight ×100.
2.4. CD163 Immunoassay
The blood samples were centrifuged at 1200 g for 10 min at 4°C, and serum was stored at −80°C until the assays. Serum CD163 levels was measured using enzyme-linked immunosorbent assay kits (R&D Systems, Wiesbaden, Germany) following the manufacturer’s guidelines.
2.5. Histological and Morphometry
2.5.1. Histological Quantification
Formalin-fixed, paraffin-embedded liver sections were cut at a 6 m thickness and then stained with Masson’s trichrome (Masson) . Images were obtained using NanoZoomer Digital Pathology system (Hamamatsu, Japan). The collagen density was quantified using Image ProPlus analysis system 7.0.1 (no. 41N70000-60555, Media Cybernetics, USA) at 100 × magnification. The data were expressed as the one-ten thousandth of collagen (the ratio of collagen area per total analyzed field area %). Values are expressed as the average of ten fields taken from each section.
2.5.2. Immunohistochemistry for Localization and Quantification of iNOS and HO-1
For immunohistochemical analysis, sections were incubated with rabbit polyclonal antibody against iNOS (1 : 500 dilution, sc-8310, Santa Cruz Biotechnology) or HO-1 (1 : 200 dilution, sc-10789, Santa Cruz Biotechnology). Staining was visualized using avidin-biotin peroxidase immunostaining kit with diaminobenzidine (Boster, Wuhan, China). The mean optical density (OD), positive staining area (AP), and observed area (AT) were determined with Image ProPlus 7.0.1 at magnifications. The levels of iNOS and HO-1 were calculated by the formula . The average of ten random fields generated a single data for statistic analysis .
2.6. Effect of Salvianolic Acid B on Isolated Portal Perfused Rat Livers with Chronic Hepatitis
The isolated portal perfusion system was performed with controlled velocity as described previously [20, 21]. At , , , and , the perfuse velocity was chosen 3935.50, 4720.63, 4753.35, or 5164.16 (L/min), respectively [20, 21]. Phenylephrine hydrochloride was determined as , , , and mol/L, respectively, to constrict portal veins . After the phenylephrine constriction, Sal B (– mol/L) was added into the recirculating perfusate. Dose-response curves were obtained from the Sal B concentrations and the changed percentage of the perfused pressure from the baseline of phenylephrine constriction.
2.7. Statistical Analysis
All data are expressed as mean ± S.E.M. Comparisons between groups were performed using Student’s t-test or Mann-Whitney. Significant differences were established at the 0.05 level. The equation, the EC50 with its 95% confidence intervals of Sal B, and the area under the curve (AUC) of Sal B were analyzed using GraphPad Prism 4 (GraphPad Software). The EC50 of Sal B was regressed with the durations (0, 28, 56, and 84 days) and serum CD163 levels in the progression of chronic hepatitis, and the AUC of Sal B () was regressed with the amounts of existed iNOS or HO-1 from immunohistochemical staining and the serum CD163 levels .
3.1. General Characterization of Rats
The model of rat PH was confirmed by ascite levels, organ index, and serum biomarker levels (Figure 2). The exuded liquid ratios significantly elevated from to () as the progression of chronic hepatitis (Figure 2). Hepatic indexes were the lowest at , the highest at , and reduced at and gradually (Figure 2). The splenic or renal indexes increased gradually from to (Figure 2). Serum ALT and AST levels increased from to , then relived at and . Serum ALP levels increased from to , then relived at . Serum Alb levels decreased from to , then relived at and (Figure 3).
3.2. Portal Pressure and Serum CD163 Levels
3.2.1. Portal Pressure
The portal pressure in vivo significantly increased from to () as the procession of chronic hepatitis (Figure 4).
3.2.2. Serum CD163 Levels
CD163 is a biomarker of the activated macrophages in PH. The serum CD163 levels were increased gradually from to () as the procession of chronic hepatitis (Figure 4).
3.3. Pathological Changes and Morphometry
3.3.1. Pathological Changes
The hepatic pathological changes induced by CCl4 were evaluated by Masson-stained sections. At , the liver showed normal hepatic architecture, and the collagen only normally distributed at the portal areas and around vessels (Figure 5(a)). At , the hepatic fatty degenerations and cellular swellings were obviously observed, and the hepatic sinusoid was severely narrowed without obvious collagen (Figure 5(b)). At , the hepatic fibrosis was observed and the collagen increased and mainly deposited in lobules. The relived enlarged hepatic cords led to the hepatic sinusoid widen obviously. Some deposited collagen in interlobular had extended into and separated lobules incompletely, thus the directions of blood flow were not changed in hepatic sinusoid (Figure 5(c)). At , the hepatic cirrhosis was evident. The lobules were completely destructed by deposited collagen and the formation of pseudolobules was observed, so the directions of blood flow were completely changed in hepatic sinusoid (Figure 5(d)).
3.3.2. Collagen Ratio
Quantification of Masson staining by morphometry analysis showed that collagen ratios were increased along with the progression of chronic hepatitis (Figure 6).
3.4. Localization and Quantification of Synthases
3.4.1. iNOS Cellular Localization
The iNOS positive cells were the hepatocytes and scattered stellates in the lobules of the normal rats at (Figure 5(e)). The iNOS expression was reduced in the scattered hepatocytes and mainly observed in stellates cells in the lobules of the rats with chronic hepatitis at (Figure 5(f)). The expression of iNOS was completely quenched in the hepatocytes; the positive cells were the macrophages in the portal triads and the stellates in the lobules at (Figure 5(g)). The main thick positive cells were the macrophages in the fibrous interval pseudolobules around vessels and the stellates with thin granules in the lobules at (Figure 5(h)).
3.4.2. iNOS Quantification
The iNOS-IHC OD per volume (Figure 6) in the portal triads of the rats with chronic hepatitis was significantly increased at (2-fold), (1.5-fold), and (3-fold) compared with that at , respectively, (); these were decreased at and increased at compared with that at , respectively (); so did increased that at compared with that at ().
3.4.3. HO-1 Cellular Localization
The main HO-1 positive cells were the hepatocytes near the central vein and the scattered stellates in the lobules, but the hepatocytes next to portal triads were absolutely negative in the normal rats at (Figure 5(i)). Besides of the thinner granules in the hepatocytes, the main positive staining cells were the stellates in the lobules at (Figure 5(j)). The thin granules have completely disappeared in the hepatocytes, while the positive cells were the macrophages in portal triads and the stellates in the lobules at (Figure 5(k)). The main thick positive cells were the macrophages in the fibrous intervals out pseudolobules at (Figure 5(l)).
3.4.4. HO-1 Cellular Quantification
The total HO-1-IHC OD per volume (Figure 6) in the rats with chronic hepatitis was significantly increased at (1.6-fold), (2-fold), and (3-fold) compared with that at , respectively (); these at and significantly increased compared with that at , respectively (); so did that at compared with that at ().
3.5. Salvianolic Acid B Reducing PH
3.5.1. Dose-Effective Relation for Relaxing Portal Vein
At , Sal B constricted portal veins of normal rats (Figure 5(m)), the equation was / (, ); the EC50 with its 95% confidence intervals was (–) mol/L. At (Figure 5(n)), (Figure 5(o)), and (Figure 5(p)) of the progression in the rats with chronic hepatitis, Sal B relaxed portal veins, the equations were / (, ), / (, ), and / (, ), respectively; the EC50 with their 95% confidence intervals were (–) mol/L, (–) mol/L, and (–) mol/L, respectively.
3.5.2. Time-Effective Relation with Pathological Progression
The liner regressive equation was (, ) from the EC50 ( mol/L) of Sal B to the durations ( (hours); , 28, 56, and 84 days) of chronic hepatitis progression. So did the equation was (, ) from CD163 levels to the durations.
3.5.3. Salvianolic Acid B-AUCs Correlated with Existed iNOS
The liner regressive equation was (, ) from the AUCs of Sal B to the iNOS-OD/V (%) in portal triads at , , , and in the progression of CCl4-induced chronic hepatitis.
3.5.4. Salvianolic Acid B-AUCs Correlated with Existed HO-1
The liner regressive equation was (, ) from the AUCs of Sal B to the HO-1-OD/V (%) in portal triads at , , , and in the progression of CCl4-induced chronic hepatitis.
3.5.5. Salvianolic Acid B-AUCs Correlated with Serum CD163 Levels
The liner regressive equation was (, ) from the AUCs of Sal B to the serum CD163 levels at , , , and in the progression of CCl4-induced chronic hepatitis. It was (, ) from the AUCs to the serum CD163 levels at , , and in the progression of CCl4-induced chronic hepatitis.
It was demonstrated in the present study that Sal B relaxed portal veins in IPPRLs of CCl4-induced chronic hepatitis. Its mechanisms are related to the inhibition of oxidative stress from macrophages and the increase of NO bioavailability or CO potency in portal triads. Sal B is the most active antioxidant extracted from Danshen and has obvious effects for liver fibrosis, chronic hepatitis, or PH in clinic .The mechanisms responsible for the protective effects of Sal B in PH remain unclear.
It has been reported that the portal resistance is mainly located at the terminal portal venules (TPV) in portal triads . The activated macrophages release vasoactive substances concomitantly and increase the perfusion resistance . Accordingly, we have previously demonstrated that the macrophages out lobules express more iNOS, produce more NO, and generate ONOO− to further reduce NO bioavailability and aggregate PH . The HO-1/CO activation decreases iNOS expression, enhances antioxidative effect, and upregulates extracellular superoxide dismutase (ecSOD) . The local ecSOD could scavenge superoxide and block ONOO− generation . Therefore, macrophage-derived NO or CO in portal triads was considered as the most effective target. Sal B, a molecule from medical plants  for PH [4–6, 20, 21, 25], has benefits to elevate NO bioavailability and to maintain CO potency , and to inhibit oxidation , especially in macrophages . We hypothesized that Sal B relaxes portal veins through anti-oxidative effects on NO or CO potency.
In present study, PH model in rat was replicated by CCl4-induced chronic hepatitis and was confirmed with increased portal pressure and pathological changes, such as the peritoneal exudation, enlarged hepatic indexes, serum hepatic biomarker levels, collagen deposition, and pseudolobule formation [1, 2]. The serum CD163 levels increased as the procession of chronic hepatitis, indicating the activation of macrophages, which was consistent with the PH patients . We found Sal B reduced PH as a candidate from a medical plant for PH patients. Sal B increased the portal pressure of the IPPRLs at physiological status and reduced the PH of the IPPRLs at chronic hepatitis status in this study. EC50 of Sal B relaxation was positively correlated with the duration of CCl4-induced chronic hepatitis, indicating the action of Sal B which was pathological dependent. Our results demonstrated that increased iNOS or HO-1 levels in the macrophages infiltrated in portal triads are involved in the mechanism of Sal B relaxation. The existed levels of iNOS or HO-1 in lobules disappeared gradually, these in portal triads strengthened continuously along with the progression of CCl4-induced chronic hepatitis, especially in the infiltrated macrophages. We also reported here that iNOS and HO-1 levels in portal triads are correlated positively with the AUCs of Sal B for reducing PH.
The IPPRL was used in this study to evaluate the effect of Sal B on PH. The hepatic artery was ligated to ensure that the portal resistance originated mainly from the smooth muscle cells in terminal portal venule (TPV) and the sphincter-like endothelia at hepatic sinusoid inlets . In PH rodents, the TPVs were the major resistance in portal microcirculation without enough collateral (like pre-TPV) or sinusoidal (post-TPV) networks to compensate a blood pressure increase . Furthermore, the infiltrated activated macrophages in portal triads were next to TPVs in the rats with oxidative chronic hepatitis (Figure 7 inserted micrographs). Sal B relived endothelin-induced elevated portal pressure in physiological rats  or mice ; these did not agree with the data in this research that Sal B increased further phenylephrine-induced elevated portal pressure in the rats without chronic inflammation [6, 20, 21]. It suggested that the macrophages infiltrated in portal triads being the indirect cellular targets of Sal B to reduce PH in the rats with chronic hepatitis. There were at least four possible pathways for Sal B decreasing PH from oxidative chronic hepatitis (Figure 7). (1) NO signal: Sal B inhibited oxidative stress of activated macrophages , blocked ONOO− generation , rescued iNOS activity from the inactivation by nitrate modification , and consequently increased local NO level to relax the TPV. Especially the AUC of Sal B for reducing PH correlated positively with the existed level of iNOS from the macrophages [8–11, 23]. Sal B relaxed indirectly portal vein via restoring NO bioavailability . (2) CO signal: Sal B increased the expression of HO-1 from activated macrophages  and elevated local CO level to dilate TPV [8, 17]. Especially the AUC of Sal B for reducing PH correlated positively with the existed level of HO-1 from the macrophages in the portal triads. Meanwhile, HO-1-derived bilirubin directly inhibits NADPH oxidase and increases ecSOD and then decreases superoxide production and ONOO− formation . (3) EcSOD protection: Sal B might indirectly upregulate ecSOD expression, which converts superoxide to hydrogen peroxide and blocks ONOO− generation from NO [11, 30]. Then the hydrogen peroxide could enhance iNOS, HO-1, and ecSOD expression itself to against the vicious cycle in PH. (4) Calcium signal: being considered as a cardiovascular protective agent , Sal B acted on TPV endothelia and smooth muscles. On human endothelia, Sal B activated transcription factor 4 or 6, consequently regulated upwards glucose-regulated protein 78, to protect the cellular damage from oxidative stress ; Sal B suppressed JAK/STAT1 activation in endothelia to relive vessel inflammation . On human vascular smooth muscle, Sal B limited calcium channel to decrease Ca2+ influx . It’s a challenge that the exact mechanism of Sal B actions from the physiological constriction switches to pathological relaxation. The clinical aspects of heme oxygenase hinted its pharmacological actions in pathological status . Further research on Sal B mechanisms might go on the way of systems biology .
Sal B for reducing PH might be used to explain the actions of medical plants in Chinese prescription for the ascitic patients with chronic hepatitis. It is an interesting clue to discover more effective candidates depending on the macrophage iNOS or HO-1 in portal triads, at least partly. Consequently, Sal B or its derivative might be exploited as a candidate to increase NO bioavailability or CO potency, especially from free radical damages in inflammatory diseases.
X. Zhao and H. Jia have the same contribution in this research work.
This work was supported by the Chinese National S&T Special Project on Major New Drug Innovation (2011ZX09307-002-01, 2012ZX09301002-001), National Natural Science Foundation (No. 30873464), and Ministry of Education Science Foundation (108019) of China.
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