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 joint 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 joint 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. We 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, natural ACTH did not significantly affect the expression of them expect NR3C1, a receptor of glucocorticoid within cells, we speculated that it has permissive effect on glucocorticoid. DXM upregulated the expression of ANGPTL4 and downregulated the expression of PDP1, PDHA1, NR3C1 and VDR, which reflected that DXM might have negative effect on the metabolism of sugars, lipids, proteins and osteocytes (28, 29) while playing the role of anti-inflammation. In inflammatory-related genes, natural ACTH did not significantly affect the expression of IL1RN and IL10, and upregulated the expression of NR1H2 and IL37 while down regulated the expression of NLRP3. As for DXM, it upregulated the expression of IL1RN and IL10 and did not significantly affect the expression of the others. This might suggest that ACTH and DXM had different mechanisms of anti-inflammatory in hepatic acute phase and immune responses (30, 31). In phagocytosis-related genes, natural ACTH did not show a significant impact on the expression of genes while DXM upregulated all of them, which might connect to the function of engulfing apoptotic cells like MERTK (32), innate resistance to pathogens and inflammatory reactions such as PTX3 (33) and FcγRIIIA (34). In addition, we also investigatived some genes that may be involved in hormone and its receptor pathways like classic receptors of ACTH on macrophages MC2R and MC3R, a kind of proto-oncogene BCL6 (35), DNA damage induced transcription protein DDIT4 (36), a transcriptional coactivator for steroid receptors and nuclear receptors PGC–1α (37), an intracellular signal transducer and transcriptional modulator SMAD3 (38) and a ligand for tyrosine-protein kinase receptors GAS6 (39). After testing and analyzing the genes above, we found that natural ACTH had a similar therapeutic effect as dexamethasone while did not have a major impact on the most of the genes’ expression as the same time. It seemed that natural ACTH was 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.