Structural Changes and Detection of Liver Fibrosis–Related Protein Levels in Coculture of Alveolar Echinococcosis-Protoscoleces and Human Hepatic Stellate Cells


 BackgroundEchinococcus multilocularis is a causative agent of human alveolar echinococcosis (AE). AE leads to cirrhosis in several organs, such as the liver, triggering severe conditions, including hepatic failure and encephalopathy. The main purpose of this study is to explore the interaction between treated hepatic stellate cells and AE-protoscoleces (AE-PSCs). The results of this study will be provided experimental basis for revealing the mechanisms of hepatic fibrosis after AE infection.MethodsWe investigated the role of alveolar echinococcosis-protoscoleces (AE-PSCs) in liver fibrosis and structural changes and liver fibrosis-related protein expression in a coculture of PSCs and human hepatic stellate cells (HSCs). Structural changes were detected by transmission electron microscopy, whereas liver fibrosis-related proteins, collagen I, alpha-smooth muscle actin, and osteopontin levels were measured by western blotting and enzyme-linked immunosorbent assay. ResultsPSCs exhibited morphological changes, specifically changes in shape, and showed slight changes in the cytoplasmic membrane, whereas structural modifications were observed in HSCs. Additionally, western blotting and enzyme-linked immunosorbent assay revealed that PSCs treated in vitro with HSC-LX2 showed significantly increased collagen-Ⅰ, α-smooth muscle actin, and osteopontin expression levels after 3–4 days of incubation in a coculture system. AE-PSCs induced liver fibrosis by inducing extracellular matrix expression and HSC activation.Conclusions﻿These results provide insight into the pathogenesis of echinococcosis- induced hepatic fibrosis and introduce therapeutic targets and diagnostic criteria for managing echinococcosis-dependent liver fibrosis.


Background
Alveolar echinococcosis (AE) is one of the deadliest human infections caused by Echinococcus multilocularis (E.m) [1][2] and is prevalent in most of the northern hemisphere [3][4]. Epidemiological studies have shown that AE is common in central Asia including areas of Kyrgyzstan, Kazakhstan, and Western China [5][6]. Its adult worms are harbored in the small intestine of de nitive hosts (such as wolves, foxes, dogs, and cats). The eggs are released in the feces of de nitive hosts. Humans become infected by eating and drinking egg-contaminated food and water. Oncospheres in the eggs are incubated in the intestines of humans, after which they migrate to the liver and live as parasites. Next, they develop into microvesicles, producing large amounts of protoscolex (PSC) in the liver [7]. These PSCs invade the Page 3/18 liver and trigger hepatic brosis. Hepatic cells at this phase are damaged by the toxic products of PSCs and their in ltration, making surgical resection di cult, and secondary infections typically occurred [8].
Patients with AE can remain asymptomatic for up to 15 years [9]. AE is often delayed when a patient with AE reports to the hospital. The parasite infection is fatal in 94% of infected people, particularly in untreated and undiagnosed patients [10][11]. Surgery and chemotherapy signi cantly improve the diagnosis of AE but have limited effects. It is impossible to completely remove the tumor because of its growth. Therefore, understanding the mechanism underlying the interactions between parasites and humans and the pathogenesis of this disease is necessary to develop treatments for echinococcosisinduced liver damage.
Hepatic stellate cells (HSCs), which are the major cells responsible for forming extracellular matrix (ECM) proteins during liver cirrhosis, are found in the Disse space area and act as a major storage site for vitamin A. They are stimulated in response to growth factors, in ammatory stimuli, and, in the case of liver damage, oxidative stress. For example, damaged liver cells, resident phagocytic cells, in ltrating in ammatory cells, aggregated platelets, and Kupffer cells can activate HSCs. Activated HSCs differentiate into muscle broblasts, which express α-smooth muscle actin (SMA) to accelerate the cirrhosis process. Under pathological conditions of cirrhosis, HSCs lose their retinoid and synthesize a large mass of ECM components, including collagen, proteoglycans, and glycoproteins [12]. In addition, they undergo proliferation, migration, and increased ECM generation, including collagen-forming bers, bronectin, and proteoglycans, which lead to septa formation in the chronically damaged liver [13]. Moreover, an imbalance in the collagen ber formation process can cause brosis [14]. Hepatic parasitic brosis caused by AE-PSCs is a host response associated with immune cell in ltration that activates HSC differentiation into broblasts [15]. Therefore, identifying the mechanisms underlying AE-induced liver cirrhosis may help reveal the disease pathogenesis and lead to the development of improved treatments.
In this study, AE-PSCs were cultured in modi ed media to investigate their role in liver cirrhosis. The human HSC-LX2 cell strain was cultured with AE-PSCs, and then liver cirrhosis-related proteins, such as Col-I, α-SMA, and osteopontin (OPN), were identi ed.

Animal infection and parasite
Mongolian gerbrils were infected with AE-PSCs for 7 months. The infected gerbrils with heavy infection was anesthetized with carbon dioxide. Masses of PSCs were collected from the killed jirds and washed with aseptic phosphate-buffered saline (PBS) under sterile conditions. After separating the jird tissue, the lesion tissue was mashed into small pieces using an aseptic sieve (mesh sieve, 300 µm) and passed through the sieve. AE-PSCs were washed ve times with PBS containing 100 µg/mL streptomycin and 100 U/mL penicillin. The PSCs were then placed in sediments at room temperature (25-30℃) for 5 min. Next, the tissues were passed through a sieve (mesh sieve, 100 µm) and washed ve times with PBS containing 100 µg/mL streptomycin and 100 U/mL penicillin at RT to remove debris. The viability of the PSCs was tested by staining with 0.1% trypan blue, and dead PSCs were stained as blue. Only PSCs showing > 90% viability were selected for further use [16]. PSCs were cultured in Dulbecco's modi ed Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 0.45% (w/v) yeast extract, 0.4% (w/v) glucose, 1000 µg/mL streptomycin, and 1000 U/mL penicillin at 37°C in the presence of 5% CO 2 .

Cell culture
Human HSCs were obtained from the Beijing University of Biological Sciences (Beijing, China). HSCs were preserved in DMEM containing Nutrient Mixture F-12 (Gibco) supplemented with 10% FBS. The cell culture was maintained and cultured in DMEM supplemented with 10% FBS containing 1000 U/mL penicillin G and 1000 µg/mL. When the cultures reached con uence, they were trypsinized and passaged at a ratio of 1:3.
Co-culture of HSC-LX2 and protoscoleces PSCs HSCs were plated into 40 Petri dishes (60 mm, 1.5 × 10 5 cells/plate) and divided into ve groups. Three groups were treated with PSCs and the other two groups were used as controls (HSCs only) and PSCs only. The three groups of HSCs were treated with PSCs and cultured at ratios Samples in the rst group were centrifuged, and the pellet was collected to measure the expression of Col-I, α-SMA, and OPN. The co-culture supernatant was collected for enzyme-linked immunosorbent assay (ELISA), and that of the other group was collected to analysis of structural changes.

Transmission electronic microscopy
Cultured human HSCs and PSCs were examined by transmission electron microscopy (TEM) to observe the cell morphology. PSC specimens for TEM were immersed in xative (2.5% glutaraldehyde) after washing three times with PBS. HSC specimens were washed with PBS and trypsin was added to the cultured cells. The cells were collected and immersed in xative (2.5% glutaraldehyde). Thereafter, the specimens were imaged by TEM (HT7700, HITACHI, JAPAN).

Western blotting
Extracellular proteins were extracted from cultured cell lysates with phosphatase and protease inhibitor cocktails. Protein (20 µg) was separated from each sample by 6% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene di uoride membrane. The membranes were blocked using 5% non-fatty milk in PBS containing 0.1% Tween-20 for 1 h at RT, and they were incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were washed with PBS containing 0.1% Tween-20 three times and then incubated with secondary antibodies conjugated with horseradish peroxidase for 90 min at RT. Protein bands were detected using a Bio-Rad Fluor-S MultiImager (Hercules, CA, USA). The band density was measured using Image software (version 1.53, NIH, Bethesda, MD, USA). β-Actin served as a loading control. The antibodies used in the analysis were anti-Col-I (ab90395), anti-α-SMA (ab7817), and anti-OPN (ab8448; Abcam, Cambridge, UK).
Detection of Col-, α-SMA, and OPN by ELISA Col-I, α-SMA, and OPN levels were measured using ELISA according to the manufacturer's instructions (CLOUD-CLONE CORP, CCC, USA). HSC-LX2 cells were treated with different ratios of PSCs for 24, 48, 72, and 96 h. Next, the DMEM was collected and centrifuged at 1000 ×g for 20 min and the pellet was collected. The supernatant (100 µL) was transferred to a new tube, and the levels of Col-I, α-SMA, and OPN were detected. The samples were then prepared and mixed with the standard sample in 96-well plates. The concentrations of Col-I, α-SMA, and OPN were determined by measuring the optical density at 450 nm using a spectrophotometer. Total levels of Col-, α-SMA, and OPN were expressed as ng/mL protein.

Statistical analyses
Data are presented as the mean ± standard deviation. Data were assessed using GraphPad Prism 8.0 software (GraphPad, Inc., La Jolla, CA, USA) and one-way analysis of variance followed by Dunnett's various comparisons test.

Culture of HSC-LX2
HSC-LX2 cells were cultured in DMEM for 3 days. After this period, and an increasing number of HSC-LX2 cells were observed in petri dishes. The cells also prominently developed broblastic morphology and contractile laments during incubation, as shown in Fig. 1.
HSC-LX2 promoted growth of AE-PSCs AE-PSCs were isolated and cultured with HSC-LX2 cells in DMEM for 3 days to evaluate the interaction between these cells. PSCs became highly motile, developed rapidly, and evaginated in the presence of HSC-LX2 [17]. In contrast, PSCs without HSC-LX2 grew slowly and were less developed, as shown in Figs Fig. 2A). Structural changes were analyzed by TEM on days 3 and 4, which revealed HSC-LX2 structural alterations. Lipid droplets were detected by TEM on day 0 and remained in normal human HSC-LX2 until days 3 and 4 (Figs. 4A-C). Lipid droplets were not observed in some cells, whereas they were degenerated in other cells on days 3 and 4 in PSC-treated human HSC-LX2 cells during the brosis transformation phase. This result reveals the brogenic transformation of these cells (Figs. 4D and 4E).
Furthermore, the Golgi apparatus was larger than normal (Fig. 4D). Some changes in the cytoplasm were observed on day 3, with autophagosomes forming on day 4 in PSC-treated human HSC-LX2 cells that were later removed by exocytosis (Fig. 4E). The nuclei of HSCs also exhibited structural changes. HSCs had two or more nuclei on day 3, and the nucleus began to fragmentation in HSCs on day 4 compared to in normal HSCs (Figs. 5A-E).

Effect of AE-PSCs on expression of OPN
In addition to Col-and α-SMA, OPN is a major ECM protein produced during brosis. Human HSCs with PSCs were incubated in coculture to identify the role of PSCs in OPN expression. Western blotting and ELISA were performed to measure OPN expression: OPN expression in treated HSCs was measured by western blotting (Fig. 8A).

Discussion
We evaluated the potential interactions of HSC-LX2 and AE-PSCs. Our results revealed that AE-PSCs activate HSC-LX2 and that HSC-LX2 effects secretion by PSCs in coculture. The development of cocultured PSCs with HSC-LX2 was better than that of control cells. PSCs were evaginated and showed rapid motility in coculture, whereas PSC control cultures grew slowly and invaginated on day 1. Moreover, the cytoplasmic content changed in the PSC control after continuous development for 4 days.
Cytoplasmic membrane changes were observed in cocultured PSCs. This result may have occurred because of the secretion of HSC-LX2, which affected the cytoplasmic membrane. Li et al. (2018) explained the development of PSCs in hepatocyte culture systems and observed that PSCs showed rapid motility and were evaginated in the rst days of culture. HSCs treated with PSCs did not contain lipid droplets compared to HSC controls. The number of lipid-containing HSCs decreased signi cantly during the transformation phase of liver cirrhosis, indicating that the cells underwent brogenic transformation [18]. All features resulting from treatment of HSCs with PSCs, including multiplication or disappearance of the nucleus, enlargement of the Golgi apparatus, changes in the cytoplasm, and nally, transformation of cytoplasm into autophagosomes, indicate brogenic transformation compared to the HSC controls.
HSCs contain one or more oval-shaped nucleoli. These morphological changes of the nucleoli were observed in HSC culture, which display thin, multiple, and elongated processes that extend from the cell body [19]. Activation of human HSCs results in expression of α-SMA, which is a speci c marker of activated HSCs [20].
Our results are similar to those of Budke et al., in which PSC reproduction was stimulated by bile salts in the intestine. Hepatocytes may produce bile, thereby stimulating PSC evasion after their release from cysts [21]. Our study revealed that HSCs produce Col-, which promotes the development of AE-PSCs. Previous studies also con rmed that collagen-integrated liver cells form bile salts in response to PSCs [21][22]. In this study, PSCs induced HSCs after 72 h, particularly at high ratios, compared with control cells that grew favorably and showed increased survival. This result is similar to that obtained in a previous study of the effect of Echinococcus species PCSs on the process of brosis [23]. Additionally, PSCs inhibit the spread of HSCs by directly targeting TGF-βR / [24]. Cystic echinococcosis cyst uid can inhibit the proliferation of HSCs and increase the main markers of HSCs, including Col-and α-SMA [23]. A previous study showed that PSCs stimulated HSC proliferation via Col-, α-SMA, and OPN production.
HSCs are typically static but become broblasts and accumulate in the ECM when activated during liver injury [8]. Thus, activation of HSCs is an important process in cirrhosis. Col-and α-SMA increased at different ratios for different incubation periods. Previous studies also showed similar results for P1CP and 3H-Pro. Collagen synthesis and α-SMA levels increased after different incubation periods [8,15]. Our experiments con rmed that OPN expression increased in HSCs treated with AE-PSCs. These results agree with those of another report on Hh signaling in liver cirrhosis. Overall, OPN and Hh are key markers of liver cirrhosis [26].
Many studies have demonstrated the role of HSCs in the synthesis of ECM components, brosis, and cirrhosis in bovine livers infected with helminths, such as Fasciola hepatica and Dicrocoelium dendriticum. HSCs play an important role in the development of brosis and other stages of cirrhosis [23,27]. TGF-β is the main factor that activates HSC and collagen deposition, as it is necessary for the differentiation of HSCs. Cirrhosis is a common progressive pathological process that occurs after extensive liver injury. ECM deposits are characteristic features of liver cirrhosis observed after hepatic cell activation. HSCs differentiate and become the main producers of the ECM [25,27].

Conclusion
We explored the interaction between treated HSCs and AE-PSCs, which revealed that AE-PSCs induced liver brosis through HSC-producing brosis proteins (i.e., Col-, α-SMA, and OPN). These ndings may contribute to understanding the pathogenesis of parasitic hepatic brosis. Additional studies are needed to focus on the parasite and its participation in this complicated process to con rm and explain the interaction between AE-PSCs and the host. Figure 1 Morphological images of activated HSC-LX2. These images were taken after 24, 48 and 72 h.

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