CE causes infiltratration of lymphocytes and dense fibrous tissue
Sirius Red and HE staining were used to investigate histomorphology. The HE staining results showed that numerous lymphocytes were infiltrated in the liver of CE patients compared to the normal samples. In addition, Sirius Red staining revealed that a substantial amount of collagen was deposited in CE liver tissues compared to the normal samples(Fig. 1A). Moreover, mouse models were used to systematically and comprehensively study the entire process involved in the course of liver infection. Therefore, 3000 and 5000 protoscolices, respectively were injected into the glisson capsule. Within an infection period of 90 days, there was limited progression of CE vesicles at the site of injection. However, when focus was expanded to the surrounding areas as infection progressed, the results showed that CE was a prolonged and progressive disease (Fig. 1B).
Furthermore, HE staining revealed that a substantial amount of lymphocytes infiltrated around lesions in the 3000 and 5000 protoscolices groups after 30 days of infection. However, the number of lymphocytes infiltrated around the lesions continuously decreased with the progression of CE. Additionally, scattered lymphocytes and closely arranged fibroblasts could be seen around the injured areas after 120 and 150 days of infection. The HE staining results also showed that the degree of inflammatory response assumed a downward trend, and tissue congestion presented was pronounced 120 days after of infection (Fig. 1C).
On the other hand, the Sirius Red stained liver sections revealed the formation of loose collagen fibrils after 30 days of infection. Afterwards, the fibrous tissue increased, became denser and eventually formed a monolayer (Fig. 1D).
CE alters the expression of NK cells surface receptor in the liver
In order to detect the impact of CE on NK cells in the liver, gate NK1.1 + CD3 was selected as the NK phenotype (Fig. 2A). Generally, murine intrahepatic NK cells can be divided into two subsets based on the relative expression of CD49a and DX5 respectively. They include circulating conditional NK cells and liver-resident NK cells[21]. The findings showed that the proportion of liver-resident NK cells increased in both the 3000 and 5000 protoscolices groups 7 days after infection but were afterwards steadily maintained at low levels and reached the lowest point on the 90th day. Notably, a novel cell subset persisted between day 14 and 90 and this may have been due to the migration of NK cells from the spleen to the damaged parts of the liver (Fig. 2B). The bar chart in Fig. 2F was therefore drawn to clearly highlight this change (Fig. 2F).
NKG2D is a marker of NK cell activation and it triggers apoptosis in HSCs by interacting with ligands on their surface to alleviate liver fibrosis[22]. In this study, expression of NKG2D reached its peak on the 7th day but then assumed a decreasing trend except on the 60th day in both the 3000 and 5000 protoscolices groups. On the 90th day, expression of NKG2D reached its lowest point in both groups, indicating the diminished ability of NK cells to limit liver fibrosis (Fig. 2C, G). Moreover, the up-regulation of NKG2D on the 60th day may have been because the migrating NK cells were at their maximum proportion (Fig. 2F).
Previous reports showed that the expression levels of NKG2A had an inhibitory effect on NK cells[23]. In this study, it was shown that the expression of NKG2A declined in CE 7 to 21 days after infection, compared to the controls in both the 3000 and 5000 protoscolices groups. However, expression of NKG2A’s was continuously up-regulated between day 30 and 90 in both groups (Fig. 2D, H).
Moreover, TIM-3 is an inhibitory receptor to NK cells[24] and can hinder NK cell-mediated cytotoxicity [25]. In addition, it was reported that blocking the TIM-3 pathway could enhance the ability of NK cells to produce IFN-γ[26]. In the present research, it was shown that the inhibitory signal TIM-3 was gradually down-regulated between the 7th and 30th day but was up-regulated between the 60th and 90th day in both the 3000 and 5000 protoscolices groups (Fig. 2E, I).
Ce Boosts The Secretion Of Cytokines Il-15 And Tgf-β1
Previous reports demonstrated that IL-15 could impede liver fibrosis [27], while TGF-β exerted the inverse effect [28]. In this study, there were increased levels of IL-15 secretion from day 7 with maximum levels on day 30 in both groups, compared to the control. However, a downward trend in the secretion levels of IL-15 in CE was observed afterwards in both groups, although the levels were persistently higher than that of the control group during the entire duration of infection (Fig. 3A).
Similar to findings from previous studies [12] the secretion levels of TGF-β1 were continuously higher than that of the control group and reached a peak on the 60th day after infection (Fig. 3B). Given that elevated secretion of TGF-β in the liver mainly occurred between day 30 and 90 after infection, immunofluorescence was used to show that this excessive secretion was mainly derived from the macrophages in both patients and mice with CE (Fig. 3C-F).
CE induces the activation of HSCs and senescence in hepatocytes
Immunohistochemical staining revealed that α- SMA was over-expressed in the liver tissue sections of CE patients. α- SMA is a marker of activated HSCs and is mainly located around the region of focus (Fig. 4A). Activated hepatic stellate cells in turn produce excessive ECM that is deposited around vesicles hence maintaining/promoting CE-related fibrosis. In addition, senescent hepatocytes induce the activation of hepatic stellate cells[29]. In this study, liver tissues from CE patients were used to investigate the presence of senescent hepatocytes during infection. Generally, cell senescence arises due to the alteration of lysosome activity and involves the expression of senescence-associated-β-galactosidase (SA-β-Gal), which is a marker of cell senescence. Moreover, senescent hepatocytes were reported to express similar genes, including p53 (TRP53) and p21 (WAF1)[14]. The results of immunofluorescent staining revealed that markers of senescence including SA-β-Gal (Fig. 4B), p53 (Fig. 4C, D), p21 (Fig. 4E) were up-regulated during infection.
The use of inhibitors does not aggravate liver damage and can relieve the dysfunction of NK cells in CE mouse models
After using the inhibitor, ELISA results showed that only Pirfenidone and combined medication (Pirfenidone + SB525334) reduced the secretion of TGF-β in liver tissues in the CE mouse model. However, SB525334 had no such effect (Fig. 5A).
In addition, liver function was assessed by testing such markers as serum ALT, AST and direct bilirubin[30]. The results suggested that inhibitors had no detrimental effect on liver function (Fig. 5B-D).
Moreover, flow cytometry revealed changes in the expression of receptors on the surface of NK cells. In the Pirfenidone medication group, there was an increase in the expression of the activating receptor NKG2D and a down-regulation of the inhibitory receptor TIM3. There was however no effect on the expression of the inhibitory receptor NKG2A. In contrast, the SB525334 medication group showed an increase in the expression of the activating receptor NKG2D and a down-regulation of both the inhibitory receptors TIM3 and NKG2A. A combination of both drugs effectively merged the efficacy of the two inhibitors. In addition, all the three medications could up-regulate the expression of DX5. However, there was no statistical difference in the therapeutic effect of combined medication and the individual drugs (Fig. 5E). Moreover, combined medication could significantly increase the expression of proliferation index Ki67 in NK cells compared to the individual drugs (Fig. 5F).
The use of inhibitors reduces hepatocyte senescence in CE mouse models Similar results were obtained from the Western blotting technique. Pirfenidone inhibited the expression p16, p21 and p53 although SB525334 merely reduced the expression of p21 and p53. A combination of Pirfenidone and SB525334 showed better results with regard to curbing senescence in hepatocytes (Fig. 6A). In addition, combined medication could significantly decrease the expression of aging-related markers compared to single medication (Fig. 6B, C, D).
Use of inhibitors relieves CE–associated fibrosis in mouse models Collagen is a major component of the ECM and is usually degraded by Matrixmetalloproteinases (MMPs). However, during the process of fibrogenesis degradation is inhibited by Tissue Inhibitor of Metalloproteinases (TIMPs )[31]. In this study, Pirfenidone promoted the production of MMP1 and reduced the expression of TIMP1. On the other hand, SB525334 down-regulated the expression of TIMP1 but had no effect on the expression of MMP1. However, a combination of Pirfenidone and SB525334 substantially reduced the expression of TIMP1 and elevated the expression of MMP1. Based on these results, combination medicine altered the composition of enzymes and this may greatly have impacted the deposition of ECM.
Compared to the control group, combined medication was superior to each of the individual medications in reducing the production of collagen I and III. Pirfenidone had no effect on the expression of TGF-β receptors I and II. In addition, combined medication more effectively reduced the expression of TGF-β receptors I and II compared to SB525334. With regard to markers of HSCs activation, a combination of Pirfenidone and SB525334 had a more potent effect than the individual drugs in curbing the expression of α-SMA (Fig. 7).