The histological structures are the most intuitive indicators to evaluate the liver health status. PE has been reported to possess hepatoprotective effects against chemical factors (drugs, alcohol) [42,62,63], physical factors (partial hepatectomy) [64], and nutritional disorders (methionine and choline deficiency, high salt intake) that induce liver histological structure damage [50,65]. For example, PE could improve the vacuolar degeneration and steatosis of hepatocytes [42,51,64], inhibit the process of hepatocyte apoptosis and necrosis [51,66], and restore normal cellularity, including a typical polygonal morphology, normal cytoplasm and nucleus, and clear cell edges [62]. For the interstitial changes in the liver, PE reduces the dilatation and congestion of hepatic sinusoids and central veins and inhibits lipogenesis and inflammatory cell infiltration [62]. Further, PE attenuates the proliferation of fibrosis tissue, collagen, and fat deposition in the damaged liver [65,67] and inhibits pseudolobule formation [64], ultimately alleviating liver fibrosis and the fatty liver process [42,50,68]. In addition, a hepatoprotective agent with the placenta as the adjuvant and the transplantation of chorionic plate-derived mesenchymal stem cells (CP-MSCs) can also reduce hepatocyte necrosis, inflammatory cell infiltration, and collagen and fat deposition [63]. Transplantation of chorionic plate-derived mesenchymal stem cells (CP-MSCs) can also reduce inflammatory cell infiltration, improve liver collagen and fat deposition, and further reduce liver fibrosis and cirrhosis [67]. In summary, PE shows excellent hepatoprotection toward liver histological structures exposed to the above pathogenic factors.

Liver function is canonically measured by several enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), and leucine aminopeptidase (LAP). In particular, lactic dehydrogenase (LDH) is a widely accepted indicator of liver injury. In addition, serum total protein (TP), liver glycogen, bile secretion, and the metabolic capacity of the liver to indocyanine green (ICG), bilirubin (BIL), fat, and alcohol can also be used to assess the severity of liver injury [69,70,71,72]. On the one hand, PE decreases the serum levels of ALT, AST, ALP, GGT, LDH [51,62,68,73,74,75,76,77,78], total bilirubin (TBIL), cholesterol (CHOL), triglyceride (TG), and non-esterified fatty acid (NEFA), and increases hepatic phospholipid and serum TP levels [62,64,66,79]. On the other hand, PE enhances the metabolic actions of the liver to bromosulfalein (BSP), iron, and alcohol, such as facilitating the scavenging of BSP and alcohol, decreasing hepatic iron deposition, and recovering the urine iron concentration [52,53,80]. Bile promotes the digestion and absorption of fat and fat-soluble vitamins secreted by hepatocytes. PE facilitates hepatic bile secretion by promoting sphincter movement and gallbladder contraction [52,53]. Moreover, PE supplementation decreases exercise-induced serum lactate elevation, increases hepatic glycogen content [74], alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activity, and decreases the area under the curve (AUC) and the maximal concentration (C_max) of alcohol [73]. However, the level of LDH increased in serum after administering PE in a report on alcohol-induced liver injury. The reason is that LDH is widely distributed in all organs, especially in the heart, liver, and muscle. After a single administration of ethanol and PE, the level of LDH in serum cannot change significantly, and the elevation of LDH might indicate liver diseases, malignancies, heart diseases, and hematological diseases [73].

In addition, a hepatoprotective agent with the placenta as an adjuvant can also reduce ALT, AST, ALP, and TBIL levels in serum [63]. After CP-MSC transplantation in the liver, the ICG metabolism increased and returned to normal levels, and TBIL levels decreased [67]. The placenta-derived stem cells (PDSCs) uptake ICG, store glucose as glycogen, and generate urea [81]. These effects are observed either by pretreatment or incubation with PE over the entire culture period, which indicates that PE could promote PDSCs’ differentiation into hepatocytes and exhibit some hepatocellular functions. In a study, the intravenous administration of PE was superior to subcutaneous administration, which was related to drug adsorption speculatively [66]. Interestingly, PE had different effects on AST in freshly isolated and primary cultured hepatocytes. The former increased while the latter decreased [82], possibly because the isolated hepatocyte membrane was damaged, and due to the additional effect of PE on the membrane; moreover, the therapeutic effect of PE is attenuated after pasteurization [68], presumably due to the inactivation of some components by high temperatures. In conclusion, PE can improve liver function by reducing liver enzyme levels and restoring the liver metabolism, synthesis, and secretion functions of BIL, fat, and alcohol.

PE contains various antioxidant components, such as uracil, L-tyrosine, L-phenylalanine, L-tryptophan, and collagen peptides. Among them, the antioxidant activity of the mixture of uracil, L-tyrosine, and L-phenylalanine accounts for around 46% of PE, and it can effectively scavenge free radicals and decrease the malondialdehyde (MDA) level [14]. The antioxidant activity of collagen peptide accounts for around 15% of PE, and it can degrade deoxyribose and scavenge free radicals [15]. L-tryptophan exerts a potent free radical scavenging effect by inhibiting the lipid peroxidation induced by oxidative stress, and its antioxidant activity is even higher than the mixture of uracil, L-tyrosine, and L-phenylalanine mentioned above [14,16].

Multiple pathogenic factors cause an increase in ROS production, which leads to OS and hepatocellular injury. For this reason, there are a variety of antioxidant systems, especially antioxidant enzymes, in the body. The critical antioxidant systems include glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), which are said to be the body’s first line of defense against ROS [83]. Furthermore, there are particularly high concentrations of glutathione (GSH) in the liver, which buffers the redox equilibrium of the cell by undergoing oxidation or reduction reactions, according to the redox potential of the cell [84]. In addition, phase II detoxification enzymes, including heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1), are involved in detoxifying OS. HO-1 regulates the balance between free heme and bound heme to prevent the accumulation of free heme, which is essential to avert heme toxicity. Moreover, HO-1 suppresses the generation of ROS by downregulating several components of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (NOX-4), including p22 phox and p67 phox [50,85]. NQO1 is known to maintain alpha-tocopherol (vitamin E) and coenzyme Q10 in their reduced antioxidant state, making it an even more potent antioxidant to protect cells against OS [86]. The above antioxidants work together to exert antioxidant effects and maintain an oxidant–antioxidant status balance in the body.

Inflammation is an adaptive response triggered by foreign pathogens or tissue injury and involves many complex interactions between cellular and inflammatory mediators, closely associated with OS and liver fibrosis [89]. Supplementation of PE reduces the accumulation of macrophages and decreases the concentrations of inflammatory biomarkers, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 [50,51,53,62,74,75,88]. Furthermore, PE increases serum interferon-gamma (IFN-γ) [74] and immunoglobulin G2a (IgG2a) [88] levels, which suggests that PE could enhance the body’s immune function. Intercellular adhesion molecule-1 (ICAM-1) is an important adhesion molecule that mediates the adhesion reaction and leukocyte migration, usually used as an indicator of inflammation [90]. PE decreases the mRNA and protein expression of ICAM-1 and inhibits the interaction of hepatocytes with lymphocytes [75]. Moreover, immunohistochemistry revealed that ICAM-1 is expressed in the amnion and chorion of the placenta [91], suggesting that it may be involved in the inflammatory process. Several investigations have reported that PE suppresses T cell activation and proliferation, and the hepatocytes showed increased levels of anti-inflammatory factors, including IL-5, granulocyte colony-stimulating factor (G-CSF), fractalkine, IL-10, and IL-13, and decreased levels of pro-inflammatory factors such as IFN-γ, IL-1β, IL-2, IL-3, IL-12, and TNF-α. Furthermore, decreases in soluble cd40 ligand (sCD40L), FMS-like tyrosine kinase 3 ligand (Flt 3L), and granulocyte macrophage-colony stimulating factor (GM-CSF) levels were also noticed [68]. It can be seen that PE could alleviate liver inflammation. However, further studies are needed to investigate the regulatory role of PE in signaling pathways.

Apoptosis refers to the autonomous and orderly death of cells controlled by genes to maintain a stable internal environment, which is associated with various morphological and functional changes, such as cell shrinkage, chromatin agglutination, DNA fragmentation, apoptosome formation, and the expression levels of pro- and anti-apoptotic factors. Furthermore, autophagy is a process involving the phagocytosis of cytoplasmic proteins or organelles into vesicles and fusion with lysosomes to form autophagic lysosomes, which degrade the contents of the lysosomes, thereby realizing the metabolic needs of the cells themselves and leading to the renewal of some organelles. Thus, apoptosis and autophagy can reflect the situation in cell injury [92,93,94]. Annexin V is a phospholipid-binding protein and can specifically bind to the phosphatidylserine of early apoptotic cells, which is used in the measurement of cellular apoptosis [95]. Propidium iodide (PI) is a cell-impermeable dye that only stains dead cells or late apoptotic cells with damaged membranes [96]. Therefore, the combination of these two methods can better detect apoptotic cells. Moreover, cysteinyl aspartate specific proteinase-3 (caspase-3) was demonstrated to be crucial for poly(ADP-ribose)polymerase (PARP) cleavage and DNA fragmentation, which are regarded as apoptotic hallmarks [51].

PE exerts anti-apoptotic effects by reducing the DNA breaks or laddering and the number of apoptotic hepatocytes showing Annexin V- or PI-positive (Annexin V+/PI+) values, increasing B-cell lymphoma-2(Bcl-2) expression, and decreasing Bcl-2-associated X protein (Bax) and caspase-3 expression, and inhibiting the cleavage of PARP [51,75,76]. Similarly, PE increases the expression of anti-apoptotic factors Bcl-2 and Bcl-xl in liver endothelial cells [50]. LC3, which exists in the LC3-I and LC3-II forms, is a well-accepted autophagy marker involved in autophagosome formation [97]. PE inhibits drug-induced autophagy in HepG2 cells, and decreases LC3 conversion from LC3-I to LC3-II (the autophagosome marker) and the expression of autophagy-related factors, including DRAM, CHOP, P53, ATG8, cTSd, BEcN1, LAMP1, ATF4, and ATF6 [51]. Taken together, PE improves liver apoptosis and autophagy, but the effects on autophagy need further study.

Liver fibrosis represents a transitional and reversible stage between chronic hepatitis and cirrhosis, which is more often seen clinically in viral hepatitis or alcohol or fatty liver. It is characterized by the excessive accumulation of an extracellular matrix (ECM), primarily collagen (Col), within the liver that destroys the normal liver architecture [98]. Therefore, it is necessary to alleviate or prevent the process of liver fibrosis. Chemical drugs, physical damage, and nutritional disorders cause liver-inflammatory cell infiltration, ROS generation, and hepatocellular degeneration, further activating HSCs and transforming them into MFBs [98]. The activated HSCs and MFBs secrete a large number of a-SMA and various ECM, including col-I, col-III, and col-IV, which disrupts the homeostasis of collagen synthesis and degradation, resulting in the accumulation of ECM in the liver and the formation of fibrotic scars gradually [99]. This homeostasis depends on the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), which are the key factors in the degradation and remodeling of the ECM. TGF-β1 is a pivotal profibrogenic cytokine in liver fibrosis and an inhibitor of hepatocyte proliferation that can induce small mothers against decapentaplegic (Smad) 2/3 transcription and activate HSCs but is negatively regulated by Smad 7 [100,101,102,103]. In addition, the serum indexes of type III procollagen (PC-III), Col-IV, laminin (LN), and hyaluronidase (HA) can effectively reflect the condition of liver injury and fibrosis, and they are commonly used as clinical indicators to reflect liver fibrosis.

PE inhibits the TGF-β-induced expression of α-SMA, Col-I, Col-III, and Smad phosphorylation and activation to reduce collagen deposition and the fibrotic area, and it increases the activity and protein expression of MMP-9. In an in vitro study, PE decreased the expression of fibrosis-related genes, including actin alpha 2(Acta2), Col1a1, and TGF-β1 [50,65,68]. The hydroxyproline (Hyp) content could reflect collagen formation and indicate the amount and consistency of scar tissue [104]. A hepatoprotective agent with the placenta as the adjuvant can also reduce collagen deposition and the fibrotic area in the liver and decrease serum levels of HA, LN, PC-III, Col-IV, and Hyp. At the same time, the expression of fibrosis-related genes, including Col-I, Col-III, α-SMA, and TGF-β1, and the protein expression levels of Smad 2/3 and p-Smad 2/3 were decreased [63]. It could also be found that the gene and protein expression levels of Col-I and α-SMA decreased, and the activities of MMP-2 and MMP-9 increased, which can limit the synthesis and deposition of Col-I in the CP-MSC-transplanted liver [67]. Additionally, immunohistochemistry revealed that MMP-9, MMP-2, and MMP-12 were expressed in the amnion and chorion of the placenta, indicating that they have the potential to treat fibrosis [91]. In summary, PE can improve liver fibrosis and collagen deposition.