Neutrophil elastase:From mechanisms to therapeutic potential

Neutrophil elastase(NE),a major protease in the primary granules of neutrophils,is involved in microbicidal activity.NE is an important factor promoting inflammation,has bactericidal effects,and shortens the inflammatory process.NE also regulates tumor growth by promoting metastasis and tumor microenvironment remodeling.However,NE plays a role in killing tumors under certain conditions and promotes other diseases such as pulmonary ventilation dysfunction.Additionally,it plays a complex role in various physiological processes and mediates several diseases.Sivelestat,a specific NE inhibitor,has strong potential for clinical application,particularly in the treatment of coronavirus disease 2019(COVID-19).This review discusses the pathophysiological processes associated with NE and the potential clinical applications of sivelestat.

Potential Application of a Wine Extract in Skin Care: How to Benefit from the Antibacterial, Antioxidant and Elastase Inhibiting Properties

Since plant polyphenols have many beneficial properties on health, the aim of this study was to evaluate the potential use of a phenolic wine extract, a by-product of wine production, for skin care on HaCaT cells. In these studies, a significant reduction of reactive oxygen species formation in HaCaT cells and severe elastase inhibition was observed. In contrast, the wine extract caused a major increase in lipase activity. The extract showed no influence on cell proliferation, but an immunomodulatory effect on the release of the interleukins IL-6 and IL-8 was found. The phenolic wine extract demonstrated a strong activity against gram-positive and gram-negative pathogens, yeasts, and fungi. Overall, our results show that the investigated phenolic wine extract is a promising ingredient for anti-aging skin care, could contribute to the improvement of skin appearance and health, and may positively affect cellulite.

Age-related changes in seminal polymorphonuclear elastase in men with asymptomatic inflammation of the genital tract

Aim： To investigate age-related inflammatory events in the male genital tract. Methods： In a total of 4 265 randomly collected patients attending the andrological outpatient clinic of the Center for Dermatology and Andrology, University of Giessen, Germany, ejaculate volume, pH-value, sperm concentration, total and progressive sperm motility, concentration of polymorphonuclear （PMN） elastase, number of peroxidase-positive cells and fructose were measured and correlated with patient＇s age. Results： While ejaculate volume, motility and fructose all correlated negatively with age, sperm concentration, PMN elastase and the pH-value showed a positive correlation. The prevalence of male genital tract inflammation （as defined by PMN elastase 〉 250 ng/mL） and its severity increased significantly. PMN elastase did not correlate with sperm motility. Fructose as a marker of seminal vesicle function showed a significant negative relationship with the PMN elastase levels, the number of peroxidase-positive cells and sperm motility. Conclusion： The significant increases of PMN elastase levels as marker of male genital tract inflammation in older men appear to be indicative of age-related changes in local immunoregulatory mechanisms. Because there is no association of PMN elastase with sperm motility, a direct inhibitory effect of this enzyme can be excluded.

Effective extraction of elastase from Bacillus sp. fermentation broth using aqueous two-phase system

This paper presents the evaluation of an aqueous two-phase system （ATPS） for extracting elastase produced by Bacillus sp. EL31410. The elastase and cell partition behavior in polyethylene glycol （PEG）/salt systems was investigated. The suitable system for elastase extraction was PEG/KHEPO4-KEHPO4, in which elastase is mainly partitioned into the PEG-rich phase, while the cells remained in the other phase. The influence of defined system parameters （e.g. PEG molecular mass, pH, NaCl addition） on the partitioning behavior of elastase is described. The concentration of phase forming components, PEG and KHEPO4-KEHPO4, was optimized for elastase recovery by means of response surface methodology, and it was found that they greatly influenced extraction recovery. The optimal ATPS was 23.1% （w/w） PEG 2000 and 11.7% （w/w） KHEPO4-KEHPO4. The predicted recovery was about 89.5%, so this process is suggested to be a rapid and convenient method for elastase extraction.

Effects of neutrophil elastase inhibitor in patients undergoing esophagectomy:A systematic review and meta-analysis

AIM: To evaluate the benefit and safety of sivelestat(a neutrophil elastase inhibitor) administration in patients undergoing esophagectomy. METHODS: Online databases including Pub Med, EMBASE, the Cochrane Library, Web of Knowledge, and Chinese databases(Wanfang database, VIP and CNKI) were searched systematically up to November 2013. Randomized controlled trials and high-qualitycomparative studies were considered eligible for inclusion. Three reviewers evaluated the methodological quality of the included studies, and Stata 12.0 software was used to analyze the extracted data. The risk ratio(RR) was used to express the effect size of dichotomous outcomes, and mean difference(MD) or standardized mean difference was used to express the effect size of continuous outcomes.RESULTS: Thirteen studies were included in this systematic review and nine studies were included in the meta-analysis. The duration of mechanical ventilation was significantly decreased in the sivelestat group on postoperative day 5 [I2 = 76.3%, SMD =-1.41, 95%CI:-2.63-(-0.19)]. Sivelestat greatly lowered the incidence of acute lung injury in patients after surgery(I2 = 0%, RR = 0.27, 95%CI: 0.08-0.93). However, it did not decrease the incidence of pneumonia, intensive care unit stay or postoperative hospital stay, and did not increase the incidence of complications such as anastomotic leakage, recurrent nerve palsy, wound infection, sepsis and catheter-related fever. CONCLUSION: A neutrophil elastase inhibitor is beneficial in patients undergoing esophagectomy. More high quality, large sample, multi-center and randomized controlled trials are needed to validate this effect.

Effect of temperature on batch elastase production by Bacillus sp. EL31410

The production of elastase by Bacillus sp. EL31410 at various temperatures was investigated. In order to study the effect of temperature on elastase fermentation, different cultivation temperatures, ranging from 39 ℃ to 28 ℃, were evaluated in shake flask. The result indicated that 37 ℃ was best for cell growth at earlier stage; while maximum elastase activity was obtained when the cells were cultivated at 30 ℃. This result was verified by batch fermentation in 5-L bio-reactor under 37 ℃ and 30 ℃ temperature, respectively. The specific cell growth rate at 37 ℃ was higher than that at 30 ℃ during earlier stage of cultivation. The maximum value [5.5 U/(h-g DCW)] of elastase formation rate occurred at 24 h at 30 ℃ compared to 4.6 U/(h-g DCW) at 30 h at 37 ℃. Based on these results, two-stage temperature shift strategy and oscillatory temperature cultivation mode were evaluated in the next study. When compared to single temperature of 37 ℃ or 30 ℃, both two-stage temperature shift strategy and oscillatory temperature strategy improved biomass but did not yield the same result as expected for elastase production. The maximum biomass (both 8.6 g/L) was achieved at 30 h at 37 ℃, but at 42 h using two-stage temperature cultivation strategy. The highest elastase production (652 U/ml) was observed at 30 ℃ in batch process. It was concluded that cultivation at constant temperature of 30 ℃ was appropriate for elastase production by Bacillus sp. EL31410.

A novel arterial pouch model of saccular aneurysm by concomitant elastase and collagenase digestion

Background: An ideal aneurysm model of cerebral aneurysm is of great importance for studying the pathogenesis of the lesion and testing new techniques for diagnosis and treatment. Several models have been created in rabbits and are now widely used in experimental studies; however, every model has certain intrinsic limitations. Here we report the development of a novel saccular aneurysm model in rabbits using an arterial pouch that is subject to in vitro pre-digestion with combined elastase and collagenase. Methods: A segment of right common carotid artery (CCA) was dissected out and treated with elastase (60 U/ml, 20 min) followed by type I collagenase (1 mg/ml, 15 min) in vitro. The graft was anastomosed to an arterial arch built with the left CCA and the remaining right CCA, while the other end of the graft was ligated. The dimension and tissue structure of the pouch were analysed immediately, 2 or 8 weeks after operation. Findings: Ten terminal aneurysms were produced. The gross mor-phology of the aneurysm resembles the human cerebral terminal aneurysms. We have observed the following pathological changes: (1) growth of the aneurysm (mean diameter increased from (2.0±0.1) to (3.2±0.3) mm at 2 weeks, P【0.001, n=7~10); (2) thinning of the aneurysmal wall (the mean wall thickness decreased to 44% at 2 weeks), which was accompanied by significant losses of elastic fibres, collagen and the cellular component; and (3) spontaneous rupture (3 out of 9, one aneurysm ruptured 24 h after operation with the other two at 2 and 4 weeks respectively). Conclusion: This rabbit arterial pouch model mimics human cerebral aneurysms in relation to morphology and histology. In particular, this model exhibited an increased tendency of spontaneous rupture.

Improved elastase production by Bacillus sp. EL31410—further optimization and kinetics studies of culture medium for batch fermentation

An efficient culture medium producing a bacterial elastase with high yields was developed further following preliminary studies by means of response surface method. Central composite design (CCD) and response surface method-ology were applied to optimize the medium constituents. A central composite design was used to explain the combined effect of three medium constituents, viz, glucose, K2HPO4, MgSO47H2O. The strain produced more elastase in the completely optimized medium, as compared with the partially optimized medium. The fitted model of the second model, as per RSM, showed that glucose was 7.4 g/100 ml, casein 1.13 g/100 ml, corn steep flour 0.616 g/100 ml, K2HPO4 0.206 g/100 ml and MgSO47H2O 0.034 g/100 ml. The fermentation kinetics of these two culture media in the flask experiments were analyzed. It was found that the highest elastase productivity occurred at 54 hours. Higher glucose concentration had inhibitory effect on elastase production. At the same time, we observed that the glucose consumption rate was slow in the completely optimized medium, which can explain the lag period of the highest elastase production. Some metal ions and surfactant additives also affected elastase production and cell growth.

Influence of medium components on elastase production using crude sources by Bacillus sp.EL31410

A newly isolated strain EL31410, producing elastase (E.C3.4.4.7) with h igh elastolytic activity was identified as Bacillus sp. In the medium opt imizat ion, it was found that wheat bran and soybean flour hydrosate were the best crud e carbon and nitrogen source for enzyme production, respectively. Addition of co rn steep flour can affect the bacterium growth and elastase production. A fracti onal factorial design was applied to study the main factors that affect the enzy me production, and central composite experimental design and response surface me thodology were adopted to derive a statistical model for the effect of wheat bra n and soybean flour hydrosate on elastase production. The experimental results s howed that wheat bran had positive effect but soybean flour hydrosate had neg ative effect, on enzyme production. An initial concentration of 3.4%(w/v) wh eat b ran and 9.4%(v/v) soybean flour hydrosate were found to be optimal for enzyme pr oduction in batch culture. The time course of elastase production in the optimiz ed medium composition was also described.

Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa

Aim： To determine the cellular distribution of secretory phospholipase A2 （sPLA2） in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Methods： Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA2 and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. Results： Although sPLA2 was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA2 was associated with enhanced binding of annexin V-fluoroscein isothiocyanate （FITC） to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA2, disturbance of acrosome structure, and loss of vitality. Conclusion： The ability of spermatozoa to release secretory phospholipase A2 is related to the acrosomal state. Premature destabi- lization of the acrosome and loss of sPLA2 can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA2 in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Optimization of elastolysis conditions and elastolytic kinetic analysis with elastase from Bacillus licheniformis ZJUEL31410

The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows： elastolysis temperature 50 ℃, elastase concentration 1 × 10^4 U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasingelastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods.

Comparing acid steatocrit and faecal elastase estimations for use in M-ANNHEIM staging for pancreatitis

AIM To compare two tests for exocrine pancreatic function(EPF) for use in M-ANNHEIM staging for pancreatitis. METHODS One hundred and ninety four consecutive patients with acute pancreatitis(AP; n = 13), recurrent acute pancreatitis(RAP; n = 65) and chronic pancreatitis(CP; n = 116) were enrolled. EPF was assessed by faecal elastase-1(FE-1) estimation and stool fat excretion by the acid steatocrit method. Patients were classified as per M-ANNHEIM stages separately based on the results of the two tests for comparison. Independent Student's t-test, χ~2 test, Kruskal-Wallis test, Mann-Whitney U test and Mc Nemar's test were used as appropriate. RESULTS Sixty-one(52.5%) patients with CP had steatorrhoea when assessed by the acid steatocrit method; 79 (68.1%) with CP had exocrine insufficiency by the FE-1 test(χ~2 test, P < 0.001). The results of acid steatocrit and FE-1 showed a significant negative correlation(Spearman's rho =-0.376, P < 0.001). A statistically significant difference was seen between the M-ANNHEIM stages as classified separately by acid steatocrit and the FE-1. Thirteen(6.7%), 87(44.8%), 89(45.8%) and 5(2.5%) patients were placed in M-ANNHEIM stages 0,?Ⅰ, Ⅱ, and Ⅲ respectively, with the use of acid steatocrit as against 13(6.7%), 85(43.8%), 75(38.6%), and 21(10.8%) respectively by FE-1 in stages 0,?Ⅰ, Ⅱ, and Ⅲ thereby altering the stage in 28(14.4%) patients(P < 0.001, Mc Nemar's test). CONCLUSION FE-1 estimation performed better than the acid steatocrit test for use in the staging of pancreatitis by the M-ANNHEIM classification since it diagnosed a higher proportion of patients with exocrine insufficiency.

Enhanced production of elastase by Bacillus licheniformis ZJUEL31410:optimization of cultivation conditions using response surface methodology

Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cul- tures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.

Nanocellulose-Based Biosensors: Design, Preparation, and Activity of Peptide-Linked Cotton Cellulose Nanocrystals Having Fluorimetric and Colorimetric Elastase Detection Sensitivity

Nanocrystalline cellulose is an amphiphilic, high surface area material that can be easily functionalized and is biocompatible and eco-friendly. It has been used singularly and in combination with other nanomaterials to optimize biosensor design. The attachment of peptides and proteins to nanocrystalline cellulose and their proven retention of activity provide a route to bioactive conjugates useful in designs for point of care biosensors. Elastase is a biomarker for a number of inflammatory diseases including chronic wounds, and its rapid sensitive detection with a facile approach to sensing is of interest. An increased interest in the use of elastase sensors for point of care diagnosis is resulting in a variety of approaches to elsastase sensors utilizing different detection technologies. Here elastase substrate peptide-celluose conjugates synthesized as colorimetric and fluorescent sensors on cotton cellulose nanocrystals are compared. The structure of the sensor peptide-nanocellulose crystals when modeled with computational crystal structure parameters demonstrates the spatio-stoichiometric features of the nanocrystalline surface that allows ligand to active site protease interacttion. An understanding of the structure/function relations of enzyme and conjugate substrate of the peptides covalently attached to nancellulose has implications for enhancing the biomolecular transducer. The potential applications of both fluorescent and colorimetric detection to markers like elastase using peptide cotton cellulose nanocrystals as a transducer surface to model point of care biosensors for protease detection are discussed.

The activities of endothelial elastase and cathepsin G in rats at oxidative stress caused by heavy metals salts

CoCl2 introduction increased cathepsin G activity in the heart and liver as well as endothelial elastase (EEl) in kidney that indicated the development of destructive processes. CoCl2 introduction decreased EEl and cathepsin G activities in blood serum and cathepsin G in lungs. HgCl2 injection decreased EEl in blood serum, heart, liver, kidney and cathepsin G in blood serum. These decreasing of proteinases activities may be caused by cytotoxic effects of heavy metals and/or the inclusion of these proteases in the destructive processes and absence of their synthesis and/or release.

Characterization of a novel variant of the second domain of bikunin with increased leukocyte elastase inhibitory activity

The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibitory activity which previously was enhanced by mutating two amino acids, glutamine 19 and tyrosine 46 to lysine and aspartate, respectively. In this study, we tried to potentiate its inhibitory activity against leukocyte elastase. A molecular docking model of the second domain of bikunin with leukocyte elastase revealed that P5 arginine 11 was a candidate residue for a third substitution. We generated six triple point mutants using site-directed mutagenesis, compared their leukocyte elastase-inhibitory activities, and selected the most potent variant with arginine 11 substituted to serine. The IC50 values for factor XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this triple point mutant prolonged the activated partial thromboplastin time and moderately reduced leukocyte elastase-induced endothelial injury. Additionally, favorable conformations created by these mutations were speculated using the structure of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mutant of the second domain of bikunin that has potent inhibitory activities against factor XIa, factor Xa, and leukocyte elastase. This variant exhibited anticoagulant activity in plasma and suppressed endothelial cell injury.

Pterostilbene supresses inflammation-induced melanoma metastasis by impeding neutrophil elastase-mediated thrombospondin-1 degradation

Objective: Chronic inflammation plays a fatal role in tumor metastasis. Pterostilbene(PTE) is a natural dimethylated analogue of resveratrol with anticancer and anti-inflammatory activities. This study aimed to investigate the inhibitory effect of PTE on inflammation-associated metastasis and explore the underlying mechanisms.Methods: Lipopolysaccharide(LPS)-induced lung inflammation and melanoma metastasis models were established in mice. After PTE treatment for four weeks, the organ index, histological changes, proinflammatory cytokines, and the expression and activity of neutrophil elastase(NE), a biomarker of neutrophil influx in the lungs, were analysed. Additionally, direct effects of PTE on NE-induced B16 cell migration were explored in wound healing and Transwell assays, and the expression of thrombospondin-1(TSP-1) and epithelial-mesenchymal transition(EMT) markers were also detected.Results: PTE obviously attenuated the LPS-induced metastasis of circulatory B16 cells to lungs by reducing the number of metastatic nodules on the lung surfaces and the lung weight/body weight ratio. PTE treatment also significantly reduced LPS-activated increase levels of tumor necrosis factor(TNF)-a and interleukin(IL)-6 in the lungs of tumor-bearing mice. In addition, increased expression and enzyme activity of NE and decreased expression of TSP-1 were observed, and these were blocked by PTE. In vitro, PTE at concentrations without cytotoxicity also markedly suppressed NE-triggered B16 cell migration, prevented NE-induced TSP-1 proteolysis and reversed the expression of vimentin, N-cadherin and Ecadherin.Conclusion: PTE could block inflammation-enhanced tumor metastasis, and the underlying mechanism might be associated with the inhibition of NE-mediated TSP-1 degradation.

Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis

A large number of protease inhibitors have been found from leeches,which are essential in various physiological and biological processes.In the curret study,a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis,which was named HMEI-A.Primary structure analysis showed that HMEI-A belonged to a new family of proteins.HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant(Ki)of 1.69×10^(−8) mol·L^(−1).Further study showed that HMEI-A inhibited the formation of neutrophil extracellular trap(NET).These results suggested that HMEIA from the leech of H.manillensis is a novel elastase inhibitor which can suppress NET formation.It may play a significant role in blood-sucking of leeches and is a potential candidate as an anti-inflammatory agent.

miRNA-141-5p Affects the Levels of Neutrophil Elastase in Preeclampsia by Regulating MAPK1

Objective:The objective of this study was to investigate the expression levels of microRNA-141-5p(miRNA-141-5p),MAPK1 and neutrophil elastase in patients with and without preeclampsia(PE),and the relationship between miRNA-141-5p and MAPK1 with respect to the secretion of elastase by neutrophils in patients with PE.Methods:Thirty patients with PE and 30 healthy pregnant(HP)women were recruited from The Second Hospital of Shanxi Medical University,Taiyuan,China,between February 2017 and July 2018.Neutrophils were isolated from 8 mL peripheral blood samples and cultured.We recorded neutrophil count and morphology during culture.Apoptosis was detected by flow cytometry in different groups at 0,24,and 48 h.The expression levels of elastase were detected in neutrophils by enzyme-linked immunosorbent assay,whereas the expression levels of miRNA-141-5p in peripheral blood neutrophils were detected by real-time polymerase chain reaction.We used TargetScanHuman Release 7.2 to analyze the target genes of miRNA-141-5p.The expression of MAPK1 in peripheral blood neutrophils was detected by western blotting.Data were analyzed by SPSS version 21.0 software,and comparisons between groups were carried out with the Studentt test.Results:There was no significant difference between the PE and HP groups(P>0.050)with regard to age or body mass index.The weight of newborns in the PE group(2846.00±600.00 g)was significantly lower than that in the HP group(3055.00±230.68 g).The number of neutrophilic granulocytes(NGs)in blood samples from the PE group was significantly higher than that in the HP group(P=0.003).There was no significant difference between the groups with regard to morphology.Apoptosis in the PE group was delayed when compared with the HP group at different time points.TheP value of apoptosis in the PE and HP groups were respectively 0.790,<0.001 and 0.030 at 0 h,24 h and 48 h.The expression levels of miRNA-141-5p in the PE group were significantly lower than those in the HP group(P<0.050).The expression levels of MAPK1 in neutrophils from the PE group were significantly higher than those in the HP group(P<0.050)by western blot.The expression levels of elastase in neutrophils from the PE group were significantly higher than those in the HP group(P<0.050).Furthermore,the number of NGs in peripheral blood from the PE group was higher than that of the HP group;however,the levels of apoptosis were lower.The expression levels of miRNA-141-5p in NGs decreased,the expression of MAPK1 increased,and the secretion of neutrophil elastase in the NG medium increased in the PE group than those in the HP group.Conclusion:Collectively,our analysis suggested that miRNA-141-5p may be involved in the pathogenesis of PE by regulating the MAPK1 signaling pathway to activate neutrophils and increase the secretion of elastase.

Relationship between Vascular Elasticity and Spontaneous Rupture of Hepatocellular Carcinoma

Objective Spontaneous rupture of hepatocellular carcinoma (HCC) is common in Asia and Africa with unclear mechanism. In our previous study, we found that the deposition of immune complex on vascular wall and vascular injury were related to the HCC rupture. In this study, the structure of elastin around the small artery was deeply investigated to confirm our previous study. Methods Immunohistochemical technique and transmission electron microscopy were used to study 23 specimens from ruptured HCC and 30 cases of nonruptured HCC. Results The layer of elastin around the vascular wall was significant thicker in patients with ruptured HCC than that in nonruptured HCC. The proliferation of elastin, abnormal distribution of neutrophil elastase and degradation of collagen fibril were predominantly present in the specimens from ruptured HCC. The phenomenon of electron—dense deposit in the elastic lamina that represented the deposition of immune complex, and the signs of infiltrated neutrophils from bloodstream into the vascular wall that caused the vascular injury, also can be found in ruptured HCC. Since the damaged vessels could become stiff and weak, which would more prone to be splitting and results in hemorrhage and the rupture of HCC, we postulated that the preexisting of immune complex deposition and vascular injury may be relate to the ruptured HCC. Conclusion The vascular injury caused by immune complex deposition might relate to ruptured HCC. Key words hepatocellular carcinoma - rupture - elastin - elastase