The innate immune system is the main defense against microorganisms, and LPS can act as an inducer of innate immunity to activate the adaptive immune system [18]. LPS binds to the PRR TLR4 to activate MAPK and NF-κB signaling [19, 20], as well as the PI3K-AKT pathway [21], generating an inflammatory response. Consistent with this, the KEGG analysis indicated that LPS activated Rap1, PI3K-Akt, NF-κB, and MAPK signaling in LMH cells. GO analysis of the 50 DEPs between the LPS and NC groups indicated that they tended to be involved in biological processes such as the immune and inflammatory responses and responses to external stimuli such as TRAF3 and RELB. Proteins belonging to the tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family function in a variety of signal transduction pathways leading to the expression of a variety of immune receptors, including innate and adaptive and cytokine receptors [22]. TRAF3 signaling stimulates the NF-κB and MAPK pathways, both of which regulate inflammation and the inflammatory response [23, 24]. RELB, a member of the NF-κB transcription factor family, is significantly up-regulated during inflammation, which induces the formation of the RELB/p50/IκBα complex [25]. It was found that LPS caused upregulation of TRAF3 in human bronchial epithelium (16HBE) [26] and also increased RELB expression in RAW 264.7 cells [27]; our results are in agreement with these findings.
The 166 DEPs identified after andrographolide treatment, compared with cells treated only with LPS, were found to be involved in 223 KEGG pathways, with significant enrichment seen in pathways associated with metabolism and HIF-1 signaling. GO analysis indicated DEP involvement in biological processes such as steroid and carbohydrate metabolism and the negative regulation of MAPK cascades. HIF is a heterodimeric complex composed of α and β subunits, with the α subunit having two forms, HIF-1α and HIF-2α. HIF-1α is a key regulator of both cell metabolism and inflammation [28, 29], and LPS can activate the expression of HIF-1α [30]. Studies have shown that there are close relationships between HIF-1α, inflammation, and cholesterol metabolism [31–34]. Cholesterol accumulation in macrophages can augment the inflammatory response [32], promoting HIF-1 activation, and may mediate liver injury [33]. Some TCM can inhibit inflammation by interfering with HIF-1α; for example, curcumin can reduce inflammation and apoptosis by inhibiting HIF-1α expression and the total cholesterol content in macrophages [35]. Our results showed that andrographolide down-regulated the expression of HMGCS1, HMGCR, and FDPS involved in steroid metabolism. The mevalonate pathway is involved in lipid metabolism and is a key regulatory step in the de novo synthesis of cholesterol, with 3-hydroxy-3-methylglutaryl coenzyme A synthase 1(HMGCS1) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) playing key roles. HMGCS1 is a cytoplasmic enzyme upstream of HMGCR in the mevalonate pathway and can condense acetyl-CoA and acetoacetyl-CoA into HMG-CoA [36], a key enzyme in the biosynthesis of liver cholesterol. HMGCR is a rate-limiting enzyme in cholesterol synthesis, and increased HMGCR activity increases cholesterol synthesis in the liver. It has been found that inhibitors of HMGCS1 and HMGCR reduce liver cholesterol synthesis, thereby protecting against liver injury [37–39]. HMGCR appears to have a functional relationship with HIF-1α, as altering the expression of HIF-1α influences HMGCR expression in zebrafish [34], and HIF-1α can mediate the transcription of insulin-inducible gene 2 (Insig-2) leading to HMGCR degradation in the liver [40]. Another key enzyme in the mevalonate pathway, farnesyl diphosphate synthase (FDPS), is involved in the conversion of acetyl-CoA to cholesterol [41]; FDPS inhibitors, such as the bisphosphonate alendronate, reduce cholesterol synthesis and are used in the treatment of hypercholesterolemia [42, 43]. Polygala tenuifolia (PTE) down-regulates genes involved in lipid and cholesterol biosynthesis, including FDPS, to reduce lipid accumulation in an obese mouse model [44]. Our experimental results showed that andrographolide may inhibit the synthesis of cholesterol by down-regulating the expression of HMGCS1, HMGCR, and FDPS, thus inhibiting the inflammatory reaction and having a protective effect on liver injury. HMGCS1, HMGCR, and FDPS may thus be drug targets for the andrographolide inhibition of liver injury.
The MAPKs are a group of related serine-threonine protein kinases, and the MAPK cascade plays a key role in a wide variety of cellular activities, including proliferation, differentiation, apoptosis, oxidative stress, and inflammatory responses [45, 46]. Numerous studies have demonstrated a link between MAPK signaling and inflammation in many diseases [47], and the activation of the ERK1/2, JNK, and p38 pathways can increase the levels of pro-inflammatory factors, such as TNF-α, IL-1β, IL-6, and IL-8 [48, 49]. MAPK signaling is activated in LPS-induced liver injury [50–52]; thus, MAPK inhibition is closely involved in preventing and treating liver injury. While research has shown that andrographolide can inhibit inflammation through the MAPK pathway, most studies have focused on the roles of JNK, ERK1/2, and p38 in classic MAPK signaling [53, 54]. Here, the proteomic results indicated that andrographolide negatively regulates the MAPK cascade and down-regulates the expression of PBK and CAV1. PDZ-binding kinase (PBK, also known as TOPK) is a serine-threonine kinase belonging to the MAPK-kinase (MAPKK) family, that promotes phosphorylation of p38 MAPK [55], leading to the identification of p38 MAPK as a specific substrate of PBK [56] and suggesting the cell-type-dependent involvement of PBK in ERK/MAPK, p38 MAPK, and JNK signaling [57]. PBK can regulate cell survival, proliferation, growth, apoptosis, and inflammation [58, 59]. It has been observed that LPS increases PBK levels in leukemia cells while up-regulating the expression of inducible nitric oxide synthase (iNOS), suggesting that PBK may be involved in the inflammatory response or in inflammation-related diseases [60]. Paeonol-based derivatives can inhibit skin inflammation by inhibiting PBK-p38/JNK signaling pathway and down-regulating nitric oxide content in LPS-induced RAW264.7 cells [61]. Caveolin 1 (Cav1) is a structural protein of cell membranes and plays a role in regulating cholesterol distribution, inflammatory signal transduction, and other biological processes [62, 63]. Cav1 is involved in hepatocellular carcinoma and hepatocellular differentiation through its activation of MAPK signaling [64, 65], and inhibition of Cav1 is associated with the anti-inflammatory effects of some drugs [66–68]. Our experimental results showed that andrographolide may negatively regulate the MAPK cascade by down-regulating the expression of PBK and CAV1, thereby inhibiting MAPK signaling. These findings provide a new idea for andrographolide reduces inflammation through the MAPK pathway.
There is normally a balance between the production of ROS and reactive nitrogen species (RNS) and oxidant scavenging in the body, and low ROS/RNS levels are essential for cell signaling and the maintenance of cell homeostasis [69]. Oxidative stress results from an altered balance between ROS and/or RNS production and the antioxidant defense capabilities [70]. The production of ROS is an important factor in liver injury [71, 72], and ROS can affect cellular structures such as hepatocyte proteins, lipids, and DNA, leading to liver injury [73]. LPS-induced liver injury is often accompanied by oxidative stress [52, 71], and inhibition of oxidative stress and inflammation can alleviate LPS-induced liver injury [74]. Antioxidants are, therefore, considered to be promising treatments for liver injury [75]. The peroxiredoxins (PRDXs) are a ubiquitous family of antioxidant enzymes, with six different PRDXs (PRDX1-PRDX6) currently known [76]. PRDXs can catalyze the breakdown of H2O2 and alkyl peroxides to eliminate intracellularly generated ROS, thereby protecting cells against oxidative stress [77]. Andrographolide is a natural antioxidant that scavenges ROS, inhibits free radical-producing enzymes, and protects the mitochondria, thereby treating diseases caused by oxidative stress [14]. We found that andrographolide up-regulated PRDX1, PRDX4, and PRDX6 and that they formed major components in the PPI networks. It has been found that PRDX1 reduces ROS levels and inhibits apoptosis induced by NF-κB or MAPK signaling [78, 79], and PRDX1 can interact with pro-Caspase-1 (CASP1) to block the assembly of the NLRP3 (NOD-, LRR-, and pyrin-domain containing protein 3) inflammasome complex, thus acting as a negative regulator of NLRP3 inflammasome activation [80]. PRDX4 is a secreted enzyme responsible for ROS scavenging both intracellularly and extracellularly and has a protective effect in cholestatic liver injury [81]. PRDX6 is a widely expressed antioxidant non-selenium glutathione peroxidase implicated in a variety of cellular activities [82]. PRDX6 protects against neuronal death caused by oxidative stress [83], and can reduce LPS-induced renal ROS concentrations and inactivate the p38 MAPK and JNK pathways, thereby attenuating LPS-induced acute kidney injury [84]. Therefore, andrographolide may inhibit oxidative stress by up-regulating the expression of PRDX1, PRDX4, and PRDX6, protecting against LPS-induced liver damage.