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  • br Materials and methods br Results br Discussion

    2023-01-02


    Materials and methods
    Results
    Discussion Recent studies have highlighted Nanaomycin A receptor and autophagy as novel targets for the treatment of liver fibrosis [7,14]. In this study, we demonstrated that catalpol protected the rat liver from CCl4-caused injury and fibrogenesis in vivo and in vitro for the first time. In particular, inhibition of inflammation played an important role in mediating the protective effects of catalpol on CCl4-induced injury and liver fibrosis. Notably, catalpol was a potent inducer of autophagy in vivo and in vitro. Importantly, inhibition or depletion of autophagy significantly attenuated the anti-inflammatory effects of catalpol on activated HSCs, indicating the crucial role of autophagy in catalpol-induced anti-inflammation. Liver fibrosis has become the major cause for liver disease-related morbidity and mortality worldwide, which represents the chronic responses of the liver to infectious, toxic and metabolic agents, potentially leading to cirrhosis and hepatocellular carcinoma [33]. Various factors, including viral hepatitis (especially hepatitis B–D), alcohol abuse, biliary atresia and hepatotoxins, can cause liver fibrosis [10,34,35]. In clinical practice, it is traditionally treated mainly by drug therapy, interventional therapy and rehabilitation therapy. However, these traditional methods not only bring severe pain to patients, but also financially burden them heavily [30]. Therefore, the pathogenesis of liver fibrosis must be further elucidated and more effective treatment strategies should be developed. Activation of HSCs is the key step in the pathogenesis of liver fibrosis [36,37]. HSCs is the main source of ECM, a major constituent of collagen deposit in liver fibrosis. Thus, controlling the activation of HSCs can effectively prevent and even cure hepatic fibrosis [24]. Herein, PDGF-BB stimulated the activation of HSCs, which was significantly inhibited by catalpol treatment, because the proliferation of HSCs and the expressions of three key markers of liver fibrosis, α-SMA, fibronectin and α1(I)-procollagen, were significantly suppressed in vitro. Rats receiving CCl4 injection also had activated of HSCs, as suggested by the significant increase of collagen deposition and hydroxyproline level. Catalpol reversed the above process in vivo, and inhibited the activation of HSCs. From the perspective of inhibiting HSCs activation, our results are consistent with previous studies [[38], [39], [40]]. Inflammation is involved in the entire process of liver fibrosis, including induction, initiation, progression and aggravation [7,41]. Pro-inflammatory cytokines, such as IL-1β, IL-18, TNF-α, COX-2 and IL-6, are produced from hepatic macrophages in various murine models of liver injury [42,43]. A large number of inflammatory cytokines are released from hepatic macrophages, forming an inflammatory microenvironment surrounding the fibrotic liver [31,44]. Thus, inhibiting the release of inflammatory cytokines and improving the inflammatory microenvironment both contribute to the recovery of liver fibrosis. Gan et al. reported that Lycium barbarum polysaccharides alleviated CCl4-induced liver fibrosis in Wistar rats probably through inhibiting the TLRs/NF-kB signaling pathway [45]. Moreover, Oró et al. found that cerium oxide nanoparticles displayed anti-inflammatory properties against rats with liver fibrosis by markedly attenuating the inflammatory response, manifested as decreased mRNA expressions of inflammatory cytokines (TNF-α, IL-1β, COX-2, iNOS) [46]. In our study, the release of inflammatory cytokines, which was promoted by CCl4 injection, was significantly inhibited by catalpol treatment, so catalpol may be an anti-inflammatory candidate for inflammation-related liver diseases including liver fibrosis. Autophagy participates in the regulation of liver homeostasis under physiological conditions [47]. Actually, the basal rate of autophagy is essential to maintain homeostasis in the liver, and alterations of the autophagic process in liver cells lead to various liver diseases including liver fibrosis [16,48]. Accumulating evidence has verified that autophagy mediated the control of proinflammatory signaling [[49], [50], [51]]. Zhang et al. reported that activation of autophagy was prerequisite for DHA to exert anti-inflammatory effects on liver fibrosis, but such effects were significantly weakened by suppressing autophagy with pharmacological inhibitors [7]. Additionally, Lodder et al. found that autophagy-deficient macrophages enhanced the fibrogenic properties of hepatic myofibroblasts via an IL1-dependent pathway, indicating macrophage autophagy may function as a novel antiinflammatory pathway regulating liver fibrosis [52]. Unlike the results of Bai et al[14], In our study, we herein found that catalpol enhanced autophagy, and inhibition of autophagy significantly decreased the anti-inflammatory effects of catalpol in vitro. In their study, rats were intragastrically given 2 ml/kg CCl4 (mixed 1: 1 in peanut oil) twice a week for 8 weeks to induce hepatic fibrosis. Contrarily, CCl4 was given by intraperitoneal injection in our experimental design. As a well-known chemical inducer, CCl4 has been commonly used to induce liver injury or fibrosis, mostly through intraperitoneal injection instead of intragastrically. Besides, Bai et al. gave the test compound trolline intraperitoneally, whereas we administered catalpol intragastrically. Thus, when administered via different routes, CCl4 pathologically damages the liver to various extents. Specifically, CCl4 given intragastrically needs to be digested and absorbed into blood, while CCl4 administered intraperitoneally can be directly exposed to the liver. In our study, catalpol significantly activated autophagy, as evidenced by the up-regulation of beclin1, Atg5 and LC3-II but the down-regulation of P62. Likewise, Zhang et al. reported that DHA promoted autophagy and activation of autophagy was essential for the anti-inflammatory effects of DHA on liver fibrosis [7]. In conclusion, catalpol activated autophagy, and activation of autophagy was critical for catalpol to exert anti-inflammatory effects. Our findings pave the way for screening proautophagic candidates and developing proautophagic drugs to treat inflammatory diseases.