Protective effect of nitric oxide on hepatopulmonary syndrome from ischemia-reperfusion injury

Abstract

AIM: To evaluate immunological protection of nitric oxide (NO) in hepatopulmonary syndrome and probable mechanisms of ischemia-reperfusion (IR) injury in rat liver transplantation.

METHODS: Sixty-six healthy male Wistar rats were randomly divided into three groups (11 donor/recipient pairs). In group II, organ preservation solution was lactated Ringer’s solution with heparin 10  000/μL at 4 °C. In groups I and III, the preservation solution added, respectively, L-arginine or N^G-L-arginine methyl ester (L-NAME) (1 mmol/L) based on group II, and recipients were injected with L-arginine or L-NAME (50 mg/kg) in the anhepatic phase. Grafted livers in each group were stored for 6 h and implanted into recipients. Five rats were used for observation of postoperative survival in each group. The other six rats in each group were used to obtain tissue samples, and executed at 3 h and 24 h after transplantation. The levels of alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α and NO metabolites (NOx) were detected, and expression of NO synthase, TNF-α and intercellular adhesion molecule 1 (ICAM-1) was examined by triphosphopyridine nucleotide diaphorase histochemical and immunohistochemical staining.

RESULTS: By supplementing L-arginine to strengthen the NO pathway, a high survival rate was achieved and hepatic function was improved. One-week survival rate of grafted liver recipients in group I was significantly increased (28.8 ± 36.6 d vs 4 ± 1.7 d, P < 0.01) as compared with groups II and III. Serum levels of ALT in group I were 2-7 times less than those in groups II and III (P < 0.01). The cyclic guanosine monophosphate (cGMP) levels in liver tissue and NOx in group I were 3-4 times higher than those of group II after 3 h and 24 h reperfusion, while in group III, they were significantly reduced as compared with those in group II (P < 0.01). The levels of TNF-α in group I were significantly lower than in group II after 3 h and 24 h reperfusion (P < 0.01), while being significantly higher in group III than group II (P < 0.01). Histopathology revealed more severe tissue damage in graft liver and lung tissues, and a more severe inflammatory response of the recipient after using NO synthase inhibitor, while the pathological damage to grafted liver and the recipient’s lung tissues was significantly reduced in group I after 3 h and 24 h reperfusion. A small amount of constitutive NO synthase (cNOS) was expressed in liver endothelial cells after 6 h cold storage, but there was no expression of inducible NO synthase (iNOS). Expression of cNOS was particularly significant in vascular endothelial cells and liver cells at 3 h and 24 h after reperfusion in group II, but expression of iNOS and ICAM-1 was low in group I. There was diffuse strong expression of ICAM-1 and TNF-α in group III at 3 h after reperfusion.

CONCLUSION: The NO/cGMP pathway may be critical in successful organ transplantation, especially in treating hepatopulmonary syndrome during cold IR injury in rat orthotopic liver transplantation.

Key Words: Nitric oxide, Nitric oxide synthase, Immunoregulatory, Hepatopulmonary syndrome, Ischemia-reperfusion injury, Orthotopic liver transplantation

INTRODUCTION

Recent research has shown that nitric oxide (NO) plays an important physiological role as a messenger in an autocrine or paracrine form. NO can influence vascular permeability, regulate vascular tone, inhibit adhesion and aggregation of leukocytes and endothelial cells, regulate platelet function, and improve graft microcirculation, thus easing vasospasm to reduce the incidence of vascular crisis[1-5]. Therefore, in this study, we established animal models of cold ischemia-reperfusion (IR) injury in rat orthotopic liver transplantation. We were able to investigate the effect of NO on hepatopulmonary syndrome and its possible immunomodulatory mechanisms during IR injury in rat orthotopic liver transplantation by enhancing or inhibiting the NO/Cyclic guanosine monophosphate (cGMP) pathway.

MATERIALS AND METHODS

Materials

Male Wistar rats weighing 180-260 g were purchased from Shanghai Experimental Animal Centre (Chinese Academy of Sciences). L-arginine and N^G-L-arginine methyl ester (L-NAME) were from Sigma, and intercellular adhesion molecule 1 (ICAM-1) (AT-29), mouse anti-rat single cloned antibody, tumor necrosis factor (TNF)-α, polyclonal rabbit anti-rat antibody, and the avidin-biotin complex (ABC) immune staining kit were purchased from Beijing Zhongshan Biotechnology Company.

Experimental methods

Experimental design: The 66 healthy male Wistar rats were randomly divided into three groups (11 donor/recipient pairs). Liver grafts were placed in different organ preservation solutions at 4 °C and stored for 6 h, and then implanted into recipients. In group II, organ preservation solution was lactated Ringer’s solution with heparin 10  000/μL at 4 °C. In groups I and III, the organ preservation solution added, respectively, L-arginine or L-NAME (1 mmol/L) based on group II, and recipients were injected with L-arginine or L-NAME (50 mg/kg) through the penis dorsal vein in the anhepatic phase. The levels of alanine aminotransferase (ALT), TNF-α, and NO metabolites (NOx) in the serum of recipients were detected, and expression of NO synthase was examined. The expression of TNF-α and ICAM-1 was observed by triphosphopyridine nucleotide (NADPH) diaphorase histochemical and immunohistochemical staining.

Orthotopic liver transplantation: All rats were anesthetized with methoxyflurane gas. All procedures described below were approved by the Committee for Animal Care and Usage for Research. Orthotopic liver transplantation was performed according to Harihara’s three-cuff technique with minor modifications, as previously reported[6-11], in which the suprahepatic vena cava was reconstructed by the Cuff method, along with the infrahepatic vena cava (IVC) and the portal vein (PV). The bile ducts were internally stented with a polyethylene stent.

Specimen measurement: The blood samples were obtained postoperatively via the tail vein at 3 h and 24 h after perfusion, or via the PV or IVC in the recipients being killed or those undergoing liver biopsy, and then centrifuged at 3000 r/min at 4 °C for 10 min. The supernatant was rapidly frozen and immediately stored at -80 °C. NOx were determined by the improved Griess method, and TNF-α was examined according to the MTT assay. Tissue levels of cGMP were detected by radioimmunoassay, protein concentration test with application of trichloroacetic acid solution and 200 g/L SDS protein precipitation method. Samples of liver and lung tissues from rats being killed or biopsied were immediately stored in liquid nitrogen, and kept frozen at -80 °C. Histopathological sections of the grafted liver were fixed in 100 mL/L formalin and prepared with hematoxylin and eosin staining for routine light microscopy.

Triphosphopyridine nucleotide diaphorase staining: The grafted liver or lung specimens were fixed in 40 g/L paraformaldehyde, 4 g/L picric and 0.1 mol/L sodium phosphate buffer (PBS), pH 7.4, for 4 h at 4 °C. Subsequently, specimens were frozen at -80 °C until section cutting. Cryostat sections were immersed for 10 min in 0.1 mol/L PBS, pH 8.0, and were incubated for 40 min at 37 °C in prewarmed solution of 0.1 mol/L PBS, pH 8.0, 3 g/L Triton X-100, 0.5 mmol/L nitroblue terazolium, and 1.0 mol/L NADPH. After being washed in 0.1 mol/L PBS, pH 7.4, sections were dehydrated with graded alcohol. Slides were rinsed in PBS and counter stained with fast red for 2 min, and coverslips were mounted on microscopic glass slides. Areas with a positive reaction for NADPH diaphorase were stained dark blue in the cytoplasm and red in the nucleus.

Immunohistochemistry: Immunohistochemical methods were used to examined the expressions of ICAM-1 (AT-29) with mouse anti-rat single cloned antibody, and TNF-α with polyclonal rabbit anti-rat antibody by the Avidin-Biotin complex method using an ABC immunostaining kit[12]. Areas with a positive reaction were stained pale brown.

Statistical analysis

Data are presented as mean ± standard error (SE). Comparisons among different groups of samples were made by two-tailed χ² test and F test. A value of P < 0.05 was considered to be statistically significant.

RESULTS

Survival

One-week survival rate of grafted liver recipients in group I was significantly increased (group I vs group II, 28.8 ± 36.6 d vs 4 ± 1.7 d, 80% vs 20%, P < 0.01), while the survival rate in group III recipients was significantly lower, compared with that of group II (group II vs group III, 0% vs 20%, P < 0.05).

Biochemical parameters

Following orthotopic liver transplantation, serum and tissue samples were assayed for ALT, NOx, TNF-α and cGMP at 3 h and 24 h after reperfusion (Table 1). The serum levels of ALT in group I were 2-7 times less than in groups II and III (P < 0.01). The levels of cGMP and NOx in liver tissue in group I were 3-4 times higher than in group II at 3 h and 24 h after reperfusion, while in group III, they were significantly reduced compared with group II (P < 0.01). The levels of TNF-α in group I were significantly lower than those in group II at 3 h and 24 h after reperfusion (P < 0.01), while there were significantly higher in group III than group II (P < 0.01, Table 1).

Table 1 Effects of serum alanine aminotransferase, guanosine monophosphate levels in grafted liver, serum nitric oxide metabolites, tumor necrosis factor-α activities and recipient survival on augmenting/inhibiting nitric oxide pathway¹.

Group	Serum ALT (nkat/L)	cGMP protein levels in grafted (ng/L)	Serum NOx (nmol/g)	Serum TNF-α(ng/L)	Weekly survival rate (%); survival days (mean ± SE)
I					80b; 28.8 ± 36.6b
3 h	837 ± 15bf	2.1 ± 0.1bf	69.4 ± 6.7b	35.8 ± 9.7f
24 h	456 ± 53bf	2.3 ± 0.2bf	22.6 ± 3.1db	24.4 ± 4.0b
II					20; 4.0 ± 1.7
3 h	1708 ± 72	0.7 ± 0.1	17.3 ± 3.4	77.2 ± 7.0
24 h	2338 ± 140d	0.8 ± 0.1	14.3 ± 2.5	55.3 ± 6.8
III					0; 1.9 ± 1.2a
3 h	2833 ± 988b	0.5 ± 0.1b	12.0 ± 2.5a	180.2 ± 20.5b
24 h	3100 ± 253b	0.4 ± 0.1b	9.9 ± 2.4a	136.6 ± 11.8b

¹During cold ischemia-reperfusion injury in the rat orthotopic liver tansplantation.

^aP < 0.05,

^bP < 0.01 vs group II;

^dP < 0.01 vs 3 h;

^fP < 0.01 vs group III. cGMP: Cyclic guanosine monophosphate; ALT: Alanine aminotransferase; TNF-α: Tumor necrosis factor-α; NOx: Nitric oxide metabolites.

Histopathology

The pathological damage in grafted liver and recipient lung tissues was significantly reduced in group I, which revealed almost normal liver sinusoidal lobular architecture, and mild degeneration of grafted liver cells at 3 h and 24 h after reperfusion (Figure 1A and C). In group III, which received NOS inhibitor, there was extensive cloudy swelling of hepatocytes, vacuolar degeneration, nuclear shrinkage, chromatin concentration, and marginalized or fragmented apoptotic bodies. We sometimes saw coagulation necrosis at the center of the central vein, small areas of spotty distribution, periportal inflammatory cell infiltration, and intrahepatic vascular thrombosis (Figure 1B). Severe structural damage to the recipient’s lung tissues, a large amount of inflammatory cell infiltration, and intravascular thrombosis were seen at the same time (Figure 1D).

Figure 1 Hematoxylin and eosin staining and triphosphopyridine nucleotide-d staining and immunohistochemical staining for grafted liver and recipient’s lung tissues. A, C: Grafted liver tissues and the recipient’s lung tissues from group I of L-arginine revealed that the pathological damages of grafted liver and lung tissues significantly reduced after reperfusion 3 h (HE × 132, × 66); B, D: Grafted liver tissues and the recipient’s lung tissues from group III of the NOS inhibitor after reperfusion 3 h showed extensive cloudy swelling of hepatocytes, vacuolar degeneration, nuclear shrinkage, chromatin concentration, and marginalized or fragmented apoptotic body and severe structural damage to the recipient’s lung tissues, a large number of inflammatory cell infiltration, and intravascular thrombosis (HE × 33, × 66); E: NADPH diaphorase histochemistry staining of NO synthase and immunohistochemistry staining of cNOS for grafted liver tissues from group I after reperfusion 24 h (NADPH-d, × 66); F: Expressions of cNOS in grafted liver tissues were particularly significant in vascular endothelial cells and hepatocytes after 24 h in group II [immunohistochemical staining (IH), × 66]; G, H: Expressions of iNOS and ICAM-1 in grafted liver tissues were little in group I of L-arginine after reperfusion 24 h (IH × 66, × 66); I, J: Expression of ICAM-1 and TNF-α in grafted liver tissues were diffuse strong in group III of the NOS inhibitor after reperfusion 3 h (IH × 66, × 66). TNF-α: Tumor necrosis factor-α; NO: Nitric oxide; NOS: Nitric oxide synthase; NADPH: Triphosphopyridine nucleotide; ICAM-1: Intercellular adhesion molecule 1; cNOS: Constitutive nitric oxide synthase; iNOS: Inducible nitric oxide synthase; HE: Hematoxylin and eosin.

Triphosphopyridine nucleotide diaphorase histochemistry

Cells that were positive for NOS were mainly expressed in the liver and endothelial cells, and were stained dark blue in the arteries, veins and capillaries (Figure 1E).

Immunohistochemistry

A small amount of cNOS was expressed in endothelial cells in normal liver tissue after 6 h cold storage, but there was no expression of inducible NO synthase (iNOS). Expression of cNOS was particularly significant in vascular endothelial cells and hepatocytes at 3 h and 24 h after reperfusion in group II (Figure 1F), but expression of iNOS and ICAM-1 was low in group I (Table 2, Figure 1G and H). There was diffuse strong expression of ICAM-1 and TNF-α in group III at 3 h after reperfusion (Table 2, Figure 1I and J).

Table 2 Intercellular adhesion molecule 1 expression in normal, storage and grafted livers.

Groups	Portal area			Sinusoidal area
	VE	BE	IC	HC	SLC	IC
Normal liver	±	-	-	-	-	-
Storage liver (6 h)	±	-	-	-	-	-
I	+	-	-	+	+	-
II	+	-	-	++	++	-
III	+++	-	-	++++	++++	-

Immunohistochemical results: "-", no significant staining; "±", a few staining; "+", focally stained; "++", diffusely stained; "+++/++++", strongly diffusely stained. VE: Vascular Endothelium; BE: Biliary epithelium; IC: Cellular infiltrates; HC: Hepato cytes; SLC: Sinusoidal lining cell.

DISCUSSION

IR injury during liver transplantation is a complex, multifactorial process in which numerous mediators and a variety of cells interact, leading to tissue damage. IR injury includes cold preservation injury, warm ischemia injury and reperfusion injury. Cold storage and warm reperfusion after restoration of blood are unavoidable steps in liver transplantation and all grafts undergo some degree of IR injury, thus leading to primary liver graft non-function or dysfunction[13]. It is a hot issue at present and a major challenge in liver transplantation. So, which mechanisms lead to primary liver graft non-function or dysfunction? What role does the NO pathway play? How to improve quality and reduce the severity of early graft donor liver during IR injury? These are problems that need to be resolved in liver transplant surgery[14].

NO has a modulatory function and is one of many organ-specific gaseous biological messengers newly discovered in recent decades in a variety of tissues or cells. NO modulates synthesis and secretion of a variety of immune mediators such as TNF-α, prostaglandin E₂, interleukin and interferon. It affects specific and nonspecific immune functions by mediating a variety of physiological and pathological phenomena, including endothelium-dependent vasodilatation, specific inhibition of proliferation of T lymphocytes, and adhesive accumulation of platelets. It plays an important extensive regulatory role in mammals. The immune regulation of NO is still not very clear in the physiology and pathology of solid organ transplantation.

L-arginine is an amino acid from which NO is synthesized. The NO synthesis pathway mainly refers to conversion of L-arginine to NO and L-citrulline under the action of NADPH cofactor and NOS in the endothelium. NO diffuses into both the vessel lumen and wall, thereby activating soluble guanylate cyclase to produce cGMP from GTP. NOx from the cGMP and non-cGMP pathway, either closely or remotely, directly or indirectly, play the role of second messengers on intracellular or extracellular target molecules[15].

Our results demonstrated that early reperfusion rapidly depletes serum arginine, because of the explosive release of large amounts of arginase, thus leading to injury of liver parenchymal cells. The deletion of arginine decreases availability of tissue arginine with subsequent downregulation of endothelial NOS (eNOS). In contrast, it can protect liver graft against IR injury and improve the histopathological damage following liver transplantation by enhancement of arginine availability through arginase blockade, or by exogenous pathways to supplement NO donors or NO inhalation, or by endogenous pathways to induce the endogenous source downstream effector of eNOS (pharmacological treatment or ischemic preconditioning)[16-18].

The non-cGMP pathway mainly affects the function of the substrate by acting on an iron-sulfur center protein. NO as an immune regulator mainly affects immune function through the non-cGMP pathway. Our study confirmed that ICAM-1 and TNF-α are also involved in the pathogenesis during reperfusion injury in liver transplantation[19]. Although our previous studies demonstrated that cellular localization of NO varies according to the immunological status of liver transplantation, there are protective effects of hepatocyte-derived NO in the hyporesponsive status, but hepatic injury is probably triggered by overexpression of iNOS of the central lobule in the hyper-responsive state. NO has neither harmful nor beneficial effects, thus, it acts as a double-edged sword, mainly depending on its source and the experimental conditions[20]. It is generally accepted that eNOS-derived NO is cell-protective by mediating vasodilatation, whereas iNOS mediates liver graft injury after transplantation[21,22]. We showed that a small amount of eNOS was expressed in liver endothelial cells in normal liver tissue and after 6 h cold storage, but there was no expression of iNOS. Expression of cNOS was particularly significant in vascular endothelial cells and hepatocytes at 3 h and 24 h after reperfusion in group II.

We confirmed that NO plays an important protective role in organ preservation solutions by supplementing sufficient NO donors to enhance the NO/cGMP pathway. It might ameliorate function of the grafted liver and recipient’s lung in hepatopulmonary syndrome, and significantly prolong the survivals of recipients after rat orthotopic liver transplantation. The levels of cGMP and serum NOx were significantly reduced after cold storage reperfusion, but there was no loss of NOS activity, suggesting that the reduction of NOx was probably the main reason for the accelerated destructive mechanism. We confirmed that NO has a significant beneficial effect on vascular function in heart and lung transplantation by strengthening NO/cGMP pathways. Therefore, by supplementing NO donors to enhance NO/cGMP pathways, it helps to balance NO/O₂ in the tendency to generate NO. The mechanisms underlying this protection involve preservation of the sinusoidal structure and maintenance of blood flow through the hepatopulmonary microcirculation[23-26]. Our previous results demonstrated that NO of hepatocytes in the grafted liver can vary the immune status of the transplanted cells, but it is likely to have important immune protective roles for NO of hepatocytes in the hyporesponsive state[27-30]. TNF-α is a wide range of polypeptide cytokines produced by a function of the body cells. Exogenous NO can significantly inhibit the macrophages stimulated by lipopolysaccharide to produce TNF-α[31]. Strengthening NO pathways significantly inhibits production and expression of TNF-α, but NOS inhibitors can contribute to increase the expression of TNF-α synthesized.

Apoptosis or programmed cell death is an active process that is controlled by specific genes without cell dissolution. Cytotoxic T lymphocytes (CTLs) leading to apoptosis can injury the target cells for lymphocyte-mediated cytotoxicity through the perforin/granzyme B and Fas/Fas-L lytic pathways[32,33]. Apoptosis as a mechanism of cell death exists in acute rejection of liver transplantation, and it is related closely to the expression of perforin, transforming growth factor-β1 and Fas-L[34]. Thus, detecting expression of CTL-associated perforin and granzyme B genes provides two valuable markers to judge the effect of immunosuppression in acute rejection of liver transplantation[35]. After reperfusion, the recipient’s lymphocyte traffic to the graft is activated, and some infiltrating lymphocytes, hepatocytes, biliary endothelial and vascular endothelial cells are induced to apoptosis. We demonstrated that it can significantly reduce the expression of ICAM-1 in transplanted liver to enhance the NO pathway, and NOS inhibitors can increase the expression of ICAM-1 during cold IR in orthotropic liver transplantation. The results indicate that it is likely related to the induction of TNF-α in the expression of ICAM-1 during ischemia reperfusion after reconstruction of portal blood flow. However, we demonstrated that it was probably thrombin-induced expression of ICAM-1 in early reperfusion after liver transplantation. We confirmed the expression of ICAM-1 in liver transplantation during IR injury. It is likely to provide a more effective method for treatment of primary grafted liver dysfunction by the application of ICAM-1 monoclonal antibody 1A29F (ab’)2[36].

Hepatopulmonary syndrome is a disorder characterized by intrapulmonary vascular dilatation, leading to gas exchange abnormalities in the setting of liver disease. Hepatopulmonary syndrome is usually a progressive disease with worsening hypoxemia developing over time[24,25,37]. The competitive inhibitor of NOS with substrate analogs such as L-NAME can reverse the local vasodilatation associated through inhibition of NOS. Administration of L-NAME abrogates the protection furnished by preconditioning[38]. Inhibition of NO synthesis by a substance such as L-NAME leads to hypotension and an effect similar to the cytotoxic effect of endotoxins. In our study, there was both the most prominent biochemical and histological deterioration of graft liver and recipient’s lung tissues, and a much higher mortality rate in the L-NAME group (group III). We observed that high survival in the L-arginine group (group I) was significantly increased, and 1-wk survival rate was 80% (P < 0.01), and one case showed long-term survival and was sacrificed on day 94. Survival of the recipients in group III was significantly reduced. One of the most important lethal causes in hepatopulmonary syndrome is hypoxia or hypoxemia, acute respiratory distress syndrome, or acute respiratory failure. This illustrated the remote protective effects of the NO cGMP synthesis pathway on the recipient’s lungs.

In conclusion, we demonstrated the novel immunomodulation of NO and a crucial role of the NO/cGMP pathway during IR injury in rat orthotopic liver transplantation and the protective mechanisms of grafted liver and recipient’s lung in hepatopulmonary syndrome. It is likely to be an effective way to improve postoperative antithrombotic therapy and the efficacy of liver transplantation by strengthening the NO/cGMP pathway.

COMMENTS

Background

Ischemia reperfusion (IR) injury during liver transplantation is a complex, multifactorial process in which numerous mediators and a variety of cells interact, leading to tissue damage. It is a hot issue at present and a major challenge in liver transplantation. In the field of solid organ transplantation, people do not know the immune suppression or immune stimulation of nitric oxide (NO) in acute rejection, chronic rejection, or the state of the immune response with low immune function.

Research frontiers

This research contributes to the theoretical basis of liver transplantation in clinical practice. The results improve the understanding of the molecular mechanisms of IR injury in liver transplantation.

Innovations and breakthroughs

Study results demonstrated novel immunoregulatory protective effects from IR injury in hepatopulmonary syndrome, involvement of intercellular adhesion molecule 1 and tumor necrosis factor-α in the pathogenesis after reperfusion injury in liver transplantation, and cellular localization of NO varies according to the immune status of the grafted liver. The study demonstrated the key role of the NO/cyclic guanosine monophosphate (cGMP) pathway during IR injury in liver transplantation. It is likely to be an effective way to provide a new pharmacological approach for preoperative antithrombotic therapy through strengthening the NO/cGMP pathway, which greatly improves the understanding of the molecular mechanisms of IR injury in liver transplantation.

Applications

Since 2003, the center has performed 97 cases of clinical liver transplantation. Eight cases complicated by hepatorenal syndrome were successfully reversed through enhancing the NO synthesis pathway by supplementing with an NO donor (L-arginine or alprostadil) in the early preoperative period. The authors suggest that acute renal failure might be successfully reversed to avoid further deterioration of multiorgan failure by the timely and decisive use of alprostadil, or by the repeated and sustained application of high-dose furosemide.

Terminology

IR injury is a complex, multifactorial process in which numerous mediators and a variety of cells interact, leading to tissue damage during liver transplantation. Hepatopulmonary syndrome is a disorder characterized by intrapulmonary vascular dilatation, leading to gas exchange abnormalities in the setting of liver disease.

Peer review

This is a good descriptive study in which authors evaluate immunological protections of NO in hepatopulmonary syndrome and the probable mechanisms of IR injury in rat liver transplantation. The results are interesting and suggest that the NO/cGMP pathway may be critical for successful organ transplantation, especially in treating hepatopulmonary syndrome during cold IR injury in rat orthotopic liver transplantation.