It can be traced to half a century ago when natural ACTH was applied to treat acute gout. Lots of studies have shown, natural ACTH can improve the function of kidney and have lipid-lowering effect(15) when compared to traditional medicine like NSAIDs and corticosteroids. Besides, ACTH can be used to treat hypoadrenia, glucocorticoid resistance and even those who have contraindications. Currently, natural ACTH is seldom used to treat acute gout because the available formulation of ACTH in most countries bears a high cost. We conducted experiments concerning the treatment of acute gout using natural ACTH, exploring its application in the treatment of acute gouty arthritis in mice and its function of regulation in mediating THP1 cells, which is stimulated by MSU. In the animal experiment, we found high-concentration of natural ACTH, when subcutaneously injected into the mice, could effectively alleviate the paw swelling and inflammation caused by MSU crystal stimulation. Although low-concentration of natural ACTH may not ameliorate the swelling, it could still prevent the accumulation of inflammatory cells. In the meantime, the level of cortisol in mice blood did not show significant changes, indicating that the application of natural ACTH will not affect the level of cortisol in mice.
When acute gout attacks patients, macrophages within articular cavity and those transformed from monocyte existing in blood will engulf MSU, secreting pro-inflammatory factors to induce inflammation(16), and thus playing an important role in the progression and development of inflammation. When focusing on this aspect, we added natural ACTH and dexamethasone into THP1 cells which had been stimulated by MSU, finding that both of them were able to inhibit the THP1 cells when considering its phagocytic function on MSU crystal. High concentration of natural ACTH could strongly inhibit the cells, the effect of which could be similar to dexamethasone. In order to verify that the inhibitory function on THP1 cells does not specifically aimed at MSU, we used FITC-latexbeads to detect the phagocytic ability of THP1 cells. The latexbeads with FITC could be engulfed by macrophage, as detected by fluorescence microscope or flow cytometry. The results are similar to that with MSU stimulation, which confirms that natural ACTH is able to inhibit the phagocytosis of THP1 cells and further prevent the MSU-stimulated secretion of inflammatory factors.
Apart from phagocytosis, macrophage will develop into pro-inflammatory M1-type macrophage during the early period of inflammation(17). MSU crystal can stimulate the cells to generate IL-1β, IL-6, TNF-α and induce inflammatory infiltration, driving the persistence of gouty inflammation(18). Our previous results showed that natural ACTH could alleviate the inflammation in the mice paw and weaken the phagocytosis of macrophage, which may be caused by the polarization of M2-type macrophage promoted by natural ACTH as we implied. Unsurprisingly, natural ACTH inhibited the MSU-stimulated THP1 cells to secret M1-type inflammatory factors, the effect of which was equal to that of dexamethasone. In the meantime, when natural ACTH was added, Arg-1 in THP1 was expressed abundantly, suggesting that natural ACTH can inhibit inflammation by promoting M2-type polarization of THP1.
The function of immune cells depends on their metabolic activity to a large extent. Therefore, the cells need to develop metabolic adaptation so as to support their various immunological functions. Macrophage, as a crucial member participating in innate immunity, is strictly regulated by metabolic pathway and metabolites(19). We cannot ignore the role of mitochondrion, which is considered as the center for cell metabolism, in regulating immune cells. In recent years, more and more regulatory mechanisms have been gradually revealed. For example, mitochondrial reactive oxygen species (mtROS), generated alongside the process of electron transfer chain, may trigger innate immune signals and damage the cells, the extent of which depends on the volume of mtROS and when does it occur(20). Studies showed that inflammation may cause an increase of ROS in the macrophage mitochondrion and inhibit the function of oxidative phosphorylation(21–22), indicating the impaired function of mitochondrion. We implied that natural ACTH can protect the mitochondrion of THP1 cells to some extent, and carried out studies in this aspect. THP1 cells could generate much more ROS in the mitochondrion when stimulated by MSU, while natural ACTH and dexamethasone ccould inhibit the process with similar inhibitory effect. mPTP is a group of protein complex existing between the inner and outer membrane of mitochondria. It is a type of non-specific channel, playing a significant role in the survival and apoptosis of cells, and involved in various fields, such as ischemia/reperfusion, cancer, aging and neural degeneration(23). mPTP allows ions whose relative molecular mass is relatively low to permeate freely under physiological conditions. It can maintain the mitochondrial membrane potential and the balance of ions within and outside the cells by driving ATP synthase through oxidative phosphorylation. However, apoptotic signals will stimulate the mPTP to open thoroughly, making soluble matter permeate nonselectively, which then leads to the imbalance of ions and membrane potential depolarization, causing apoptosis or necrosis(24–25). mPTP can be used to detect the impairment of mitochondrional function, and thus we measured the protective function of natural ATCH on mPTP. Results showed that a great amount of mPTP existing in THP1 cells opened when being stimulated by MSU, indicating the function of mitochondrion was damaged. To the contrary, natural ACTH and dexamethasone reversed the phenomenon, and the function of natural ACTH was superior, suggesting natural ACTH is protective to mitochondrion in THP1 cells and maintains the functionality of mPTP. Meanwhile, we selected several genes, such as XDH, MPO, NOX1 and NOX3, all of which are associated with the generation of ROS to conduct qPCR test, and focused on XDH. Natural ACTH can inhibit the expression of XDH in MSU-stimulated THP1 cells, and XDH is able to turn products of purine metabolism into uric acid(26). Thus, we imply that natural ACTH can inhibit the generation of uric acid and protect the function of mitochondrion. However, more studies are needed to confirm the proposal.
When treating acute gout, patients who cannot react to colchicine or NSAIDs, or those who suffer from renal insufficiency, will always consider glucocorticoid as treatment. Although glucocorticoid are effective, it cannot be ignored that side-effects such as central obesity, infection, calcium loss, osteoporosis, diabetes, and stomach ulcers, etc.(27), will also bring inconvenience to patients. It is a big concern on how to largely avoid side-effect while receiving treatment. Here, a series of experiments found that natural ACTH was equal to dexamethasone when concerning their effect on treating acute gout. We still doubt whether there is difference existing between them when concerning the effect of treatment, and whether natural ACTH is a better choice for treating acute gout, which is also a question worth investigating. In addition to the results of the PCR array, we selectively chose some intriguing genes about metabolism, inflammation, phagocytosis and some others that may be involved during the process of exerting the anti-inflammatory effect. In metabolism-related genes, ANGPTL4 can mediate the inactivation of lipase in cells, causing hypetriglyceridemia and playing a role in lipid disorders, obesity and coronary heart disease(28). We here found that dexamethasone could significantly upregulate the transcription level of ANGPTL4, while ACTH had no evident effect. PDP1, pyruvate dehydrogenase phosphatase catalytic subunit 1, can mediate the dephosphorylation and activation of pyruvate dehydrogenase, and therefore promote the tricarboxylic acid. PDHA1, pyruvate dehydrogenase α subunit, represents the key step for entering tricarboxylic(29). The intervention of Dexamethasone here significantly down-regulated the expression of PDP1 and PDHA1, suggesting that the application of glucocorticoid disturbed the normal procedure of saccharometabolism, while natural ACTH had no such effect. NR3C1 is a receptor of glucocorticoid within cells. In the presence of natural ACTH, its expression was up-regulated, which may because it has permissive effect on glucocorticoid. However, it was down-regulated after dexamethasone intervention, which was attributed to the negative feedback. VDR is a nuclear receptor of calcitriol, and was detected to be down-regulated by dexamethasone intervention, indicating that hormones may affect the synthesis and catabolism of bones. In inflammatory-related genes, IL1RN is an antagonist of IL1 that can inhibit inflammation, and was found to be down-regulated by dexamethasone in our study. NR1H2 plays an anti-inflammatory role during the hepatic acute phase response by acting as a corepressor and inhibits the hepatic acute phase response by preventing dissociation of the N-Cor corepressor complex(30); IL-37 is a suppressor of innate inflammatory and immune responses involved in curbing excessive inflammation(31), natural ACTH up-regulated their expression while dexamethasone did not, suggesting that two drugs may have different effects in anti-inflammatory. As indispensable parts of the inflammatory response, the role of NLRP3 and IL-10 has long been known. Natural ACTH could down-regulated the expressiong of NLRP3 while had no such effect on IL-10, prompt that natural ACTH does not rely on IL-10 to exert its anti-inflammatory effect. In phagocytosis-related genes, MERTK is a kind of protein on the surface of macrophage which is able to activate macrophage and promote it to engulf apoptotic cells(32); PTX3 plays a role in the regulation of innate resistance to pathogens and inflammatory reactions(33); FcγRIIIA is a receptor for the Fc region of IgG which Binds complexed or aggregated IgG and also monomeric IgG. It mediates antibody-dependent cellular cytotoxicity(ADCC) and other antibody-dependent responses such as phagocytosis(34). Dexamethasone up-regulated all of them while natural ACTH only up-regulated the expression of FcγRIIIA, suggests that they affect cell phagocytosis in different ways. MC2R and MC3R, as classic receptors of ACTH on macrophagesm, had been confirmed by many studies to be a key factor for ACTH to exert its anti-inflammatory effect through a steroid-independent pathway(14–15).We tested the expression of them after the stimulation of natural ACTH and dexamethasone, found that both of them were down-regulated by natural ACTH, we speculate that it may be a result of some kind of negative feedback regulation which need to be further examined. In addition, we also investigatived some genes that may be involved in hormone and its receptor pathways. BCL6 is a crucial transcription factor, essential for the formation of lymphoid germinal center and maturity of antibody affinity(35), it is also a kind of proto-oncogene. It needs further confirmation in vivo whether the expression of BCL6 significantly down-regulated by dexamethasone can be explained at the cellular level. DDIT4, a type of DNA damage induced transcription protein, is able to inhibit Rapamycin complex 1 and may affect cell proliferation, metabolism, autophagy and apoptosis through mTOR pathway(36). Hypoxia can significantly raise the level of DDIT, which is able to hinder the abnormal expression of downstream apoptotic proteins, and therefore to prevent apoptosis(36). Here, application of dexamethasone significantly induced its expression while natural ACTH did not show such effect. PGC-1α is a transcriptional coactivator for steroid receptors and nuclear receptors which plays an essential role in metabolic reprogramming in response to dietary availability through coordination of the expression of a wide array of genes involved in glucose and fatty acid metabolism(37);SMAD3 is an intracellular signal transducer and transcriptional modulator activated by TGF-β and activin type 1 receptor kinases, also acts as a regulator of chondrogenesis and osteogenesis and inhibits early healing of bone fractures(38), Nactural ACTH showed no significant change while dexamethasone up and down-regulated their expression respectively, suggested that the use of dexamethasone may affect the body's normal function of physiological metabolism and healing. GAS6 is a ligand for tyrosine-protein kinase receptors AXL, TYRO3 and MER whose signaling is implicated in cell growth and survival, cell adhesion and cell migration(39). The expression of GAS6 upregulated by natural ACTH might suggest that it has the ability of protecting cells’normal function. After testing and analyzing the genes above, we believed that natural ACTH had a similar therapeutic effect as dexamethasone, while also protects cells and does not have a major impact on the body's metabolism. It seemed that natural ACTH were doing well in treating acute gout inflammation while avoiding the side effects of drugs.
To summarize, we explored the function of natural ACTH in treating acute gout, which includes alleviating the inflammation and regulating macrophage. At the same time, we also found that natural ACTH is different from corticosteroids on the level of gene transcription. Natural ACTH is an attractive therapeutic option for patients who may be problematic with NSAIDs, steroids or colchicine. Moreover, ACTH is nowadays widely available and inexpensive at least in China and most European countries(13). In our study, natural ACTH showed a lot difference from dexamethasone in terms of the transcription of inflammatory and metabolic related genes. It is still worth studying wether there is difference between natural ACTH and corticosteroids and whether natural ACTH can be used as a safe alternative to corticosteroids.