TO THE EDITOR
Non-alcoholic fatty liver disease (NAFLD) is a chronic condition characterized by a marked accumulation of lipid droplets (LDs) in hepatocytes (steatosis), which can progress to non-alcoholic steatohepatitis (NASH). Morphologically, NASH is characterized by steatosis, hepatocyte injury and inflammatory hepatocellular ballooning, which can progress into fibrosis, cirrhosis and hepatocellular carcinoma. Currently, no licensed drugs have been approved for the treatment of NASH[1,2]. Xiaoyao San (XS) is a traditional Chinese medicine used for the treatment of liver stagnation and spleen deficiency syndrome, depression, various gynaecological diseases, cancer, steatosis and more. The XS formulation consists of eight Chinese herbal medicines: Bupleurum chinense DC., Angelica sinensis (Oliv.) Diels, Paeonia lactiflora Pall., Atractylodes macrocephala Koidz., Poria cocos (Schw.) Wolf, Glycyrrhiza uralensis Fisch., Zingiber officinale Roscoe and Mentha haplocalyx Briq. at a weight ratio of 3:3:3:3:3:1.5:1:1[3-9].
In an interesting paper, Mei et al[10] investigated the hepato-protective effects of a modified XS (MXS) formula on a male rat model of NASH using light microscopy, immunohistochemistry (IHC), Western blot, and metabolomics analysis of male steroid hormone metabolites. The authors found that six-week MXS treatment suppressed steatosis, fibrosis, and inflammation markers in the NASH model. This reduction of steatosis was associated with adenosine 5’-monophosphate-activated protein kinase (AMPK) activation, restoration of the phosphoinositide 3-kinase/phosphatase and tensin homolog (PTEN) expression and upregulation of androgens. These morphological and molecular findings were linked to a significant reduction in liver weight and liver weight/body weight ratio in the MXS-treated NASH group compared to the untreated NASH group. In the next paragraphs, we discuss these findings, explain the related mechanisms, and offer further perspectives.
Reduced hepatic steatosis, fibrosis and inflammation in NASH model by MXS: Morphological and molecular evidence
Using oil-red staining for neutral lipids (a marker of LDs), Mei et al[10] reported a marked decrease in LDs in the MXS-treated NASH group compared to the untreated NASH group. LDs have specific proteins surrounding their fat core, known as perilipins. The upregulation of these proteins has been associated with enhanced hepatic steatosis in NAFLD and NASH[1,11]. Therefore, studying the expression of these proteins under MXS treatment in a NASH model using IHC and Western blot could provide interesting insights into the mechanisms involved. In agreement with Mei et al[10], two studies have reported the suppression of hepatic steatosis by XS through various mechanisms, including inhibition of the glucocorticoid receptor/perilipin-2 signaling pathway[5] and activation of the estrogen receptor α pathway in ovariectomized ApoE-/-mice[3]. Furthermore, XS decoction reduced hepatic fibrosis in rats via the transforming growth factor-β/Smad and protein kinase B/forkhead box O3 signaling pathways[4]. Moreover, Mei et al[10] reported the decreased expression of the fibrosis marker alpha smooth muscle actin (α-SMA), the inflammation marker cyclooxygenase-2 (COX-2), and lipogenesis factors, fatty acid synthase (FASN) and peroxisome proliferator-activated receptor gamma (PPARγ) in the livers of the MXS-treated NASH group compared to the untreated NASH group[10]. The findings were based on α-SMA and FASN IHC analysis and COX-2 and PPARγ Western blot analysis. However, in the study by Mei et al[10], IHC of hepatic α-SMA did not clearly show the morphology of the stellate cells, which are the main effectors for hepatic fibrosis in NASH[12]. Therefore, higher magnification imaging is needed to better visualize these cells. In addition, immunoelectron microscopy could be a valuable technique for detecting stellate cells using α-SMA immunogold labelling[11].
Mechanisms of MXS-induced reduction of hepatic steatosis in NASH: Role of androgens, AMPK and PTEN
A key finding in the study by Mei et al[10] was the significant upregulation of androgens in the MXS-treated NASH group. Mechanistically, metabolomics analysis revealed that this upregulation occurred via regulation of the steroid sex hormone-related metabolic pathway. Androgen upregulation was associated with activation of AMPK and enhanced expression of PTEN in hepatocytes of the MXS-treated group. Despite these findings, several critical questions and unresolved issues warrant further investigation: (1) What is the relationship between androgen upregulation and the suppression of hepatic steatosis by MXS in the NASH model? (2) What is the mechanism of androgen upregulation with MXS treatment? (3) How do activation of AMPK and restoration of PTEN expression by MXS treatment reduce hepatic steatosis? And (4) Are there any possible additional mechanisms for the reduction of hepatic steatosis by MXS treatment?
A growing body of evidence indicates that androgen levels are reduced in men and animal models of NAFLD, NASH and models of obesity and metabolic syndrome[13,14]. Importantly, testosterone therapy was found to reduce hepatic steatosis in men with type 2 diabetes[15] and suppress the expression of FASN. Thus, this therapy appeared to protect against hepatic steatosis in cholesterol-fed androgen-deficient mice[16]. Moreover, endogenous testosterone has been reported to alleviate hepatic steatosis in protein-restricted male rats[17]. These findings align with the study by Mei et al[10], which reports that suppression of FASN in the MXS-treated NASH group was linked to reduced hepatic steatosis. Taken together, the androgens appear to reduce hepatic steatosis by suppressing lipogenic factors such as FASN, while hepatic steatosis itself can suppress androgen, hence answering the first and second questions raised. This bi-directional relationship underscores the need for further research to fully understand the mechanisms involved.
In NAFL and AFL diseases, activation of AMPK was reported to reduce hepatic steatosis by down-regulating lipogenic gene expression (FASN, sterol regulatory element-binding protein 1c, ACC and HMGCR). Activation of AMPK also enhances the expression of fatty acid oxidation proteins involved in lipolysis (carnitine palmitoyl transferase 1, peroxisome proliferator-activated receptor-γ coactivator 1, hormone-sensitive lipase, and adipose triglyceride lipase). Furthermore, AMPK activation improves mitochondrial function and integrity[18,19]. PTEN is another key regulator in cellular functions, including signaling, lipid and glucose metabolism, as well as cell survival, tumor suppression and apoptosis. The loss of hepatic PTEN results in increased de novo lipogenesis through robust induction of sterol regulatory element-binding protein and FASN expression[20,21]. These reports support the study by Mei et al[10], who observed AMPK activation and increased PTEN expression following MXS treatment in the NASH group. This observation was associated with suppression of the lipogenic factors FASN and PPARγ and answer the third question raised. Figure 1 summarizes the various mechanisms of MXS-related hepatoprotection in the NASH male rat model, based on the study Mei et al[10] and proposed mechanisms for future research.
Figure 1 Mechanisms of modified Xiaoyao San-induced suppression of hepatic steatosis and inflammation in a non-alcoholic steatohepatitis male rat model based on the study by Mei et al[10] and proposed additional mechanisms.
NASH: Non-alcoholic steatohepatitis; COX-2: Cyclooxygenase-2; α-SMA: Alpha smooth muscle actin; FASN: Fatty acid synthase; PPARγ: Proliferator-activated receptor gamma; AMPK: Adenosine 5’-monophosphate-activated protein kinase; PTEN: Phosphoinositide 3-kinase/phosphatase and tensin homolog.
Perspective: Activation of autophagy/lipophagy in hepatocytes of NASH model by MXS treatment as possible hepato-protective mechanism
Macroautophagy (hereafter referred to as autophagy) is a prosurvival bulk degradation pathway that degrades almost all cellular components via lysosomes and is specifically upregulated upon exposure to various stressors such as oxidative stress, mitochondrial damage and LD overload[11]. In addition, autophagy selectively clears damaged organelles such as mitochondria (via mitophagy), and LDs (via lipophagy)[11,22]. Impaired autophagy or lipophagy leads to various diseases, including NAFL/NASH and metabolic syndrome; conversely autophagy activation by natural products or drugs such as rapamycin reduces steatosis in AFL and NAFL[11,22,23]. Autophagy–related gene (ATG) products are the key components of autophagy; more than 40 ATGs are found in yeast such as ATG 8 and its mammalian homolog, microtubule-associated protein 1A/1B-light chain 3 (LC3). Autophagy is initiated by AMPK activation and mechanistic target of rapamycin inhibition, resulting in the formation of Beclin1-mediated autophagosomal membranes. These membranes mature into LC3-II- mediated autophagosomes, which engulf the cellular contents, then fuse with lysosomes to be later degraded by different lysosomal enzymes[11,22,23]. Importantly, XS has been reported to exert anti-depressant effects via activation of autophagy and formation of autophagosomes in mice hippocampal and hypothalamic neurons[7,8]. Therefore, we speculate that MXS could activate AMPK-mediated autophagy upregulation in hepatocytes of the NASH model, resulting in the suppression of steatosis and the reduction of liver weight and liver weight/body weight ratio in the NASH group after six weeks of MXS treatment[10]. This is because autophagy upregulation not only clears LDs but also abnormal proteins and damaged organelles from the liver of NASH models[11,22,23]. Additionally, autophagy activation by MXS could be a possible mechanism for androgen upregulation (Figure 1) based on recent studies, indicating that autophagy enhances testosterone production by Leydig cells[24-26]. This could be an additional answer to the second question. Further research is needed to explore the proautophagic effects of MXS on the liver and testis in the NASH model.
