Livin expression promotes keratinocyte release of inflammatory mediators in psoriasis.

BACKGROUND: Psoriasis is a prevalent, long-term skin condition characterized by inflammation. Keratinocytes (KCs) are important effector cells that release inflammatory factors and chemokines to promote the inflammatory cascade in psoriasis. However, the mechanisms underlying the activation of KCs in psoriasis remain unclear. Livin suppresses apoptotic proteins and directly affects the growth and spread of cancer cells. Livin expression reportedly increases significantly in lesions of patients with psoriasis; however, its specific role in KC activation remains unknown. This study aimed to examine the impact of Livin on KC activation and the subsequent release of inflammatory mediators. METHODS: Immunofluorescence staining, reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and western blotting were used to assess Livin expression in patients with psoriasis, an imiquimod (IMQ)-induced psoriasis-like mouse model, and M5-treated HaCaT cells. To investigate the role of Livin in KCs, we performed RNA sequencing and proteomic analysis of Livin-knockdown (knockdown-HaCaT) and negative control (NC-HaCaT) cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used for enrichment analyses. Moreover, the effect of Livin expression on the release of inflammatory mediators in KCs was verified using ELISA. RESULTS: Livin expression was higher in KCs of patients with psoriasis than in those healthy controls. Livin expression in HaCaT cells treated with M5 increased significantly over time. Livin expression was higher in the skin lesions of the IMQ mouse model than in the control group. Proteomic analysis and RNA sequencing used to investigate the function of Livin in HaCaT cells revealed its potential role in mediating KC activation and inflammatory mediator release, which affected the pathology of psoriasis. CONCLUSIONS: Livin expression played an effect on KCs activation, which induced release of inflammatory mediators and up-regulation of keratin. This study provides a new effector molecule for the mechanism of inflammatory response in psoriasis.

Livin upregulation in keratinocytes of psoriasis patients to promote adhesion molecule expression.

BACKGROUND: Activation of keratinocytes (KCs) is the main pathological feature of psoriasis. KCs recruit neutrophils by releasing various antimicrobial peptides and chemokines, which is also related to the expression of KC adhesion molecules. However, the regulatory mechanism governing their expression is still unclear. Livin, an inhibitor of the apoptosis protein family member involved in proliferation and metastasis of tumor cells, is significantly increased in psoriatic lesions. OBJECTIVES: The aim of this study was to investigate the role of Livin in regulating adhesion molecule expression in KCs and release of chemokines that promote the activation and adhesion of neutrophils. METHODS: The expression of Livin in psoriasis patients, imiquimod mouse model, and the combination of IL-17 alpha, IL-22, IL-1 alpha, OSM, and TNF-α (Mix M5)-treated HaCaT cells were detected by immunofluorescence staining, RT-qPCR, and ELISA. Livin-overexpression and knockdown in HaCaT cells transfected with HIV-1-based lentiviral vectors were used to study the function of Livin using RNA-seq. Moreover, differences in the expression of HaCaT cell adhesion molecules after regulation of Livin expression and activation of neutrophils in the co-culture model were verified. RESULTS: Livin was upregulated in the KCs of psoriasis patients, imiquimod mouse model and Mix M5-treated HaCaT cells compared with the control groups. Livin in HaCaT cells might regulate the expression of adhesion molecules in KCs. CONCLUSION: Thus, Livin may be a key effector molecule that regulates the expression of adhesion molecules in KCs and promotes the activation and adhesion of neutrophils.

The Efficacy of Combination Therapy Involving Excision Followed by Intralesional 5-Fluorouracil and Betamethasone, and Radiotherapy in the Treatment of Keloids: A Randomized Controlled Trial.

Background: Combined therapy for keloids is currently recommended. Surgery is one of the main options, but the measures to prevent recurrence after excision are still being explored. Objective: The randomized controlled study aimed at evaluating the efficacy of excision followed by intralesional low concentrations of 5-fluorouracil (5-FU)(12.5 mg/mL) and betamethasone. Methods: Sixty patients were randomly assigned to three groups. Patients in group A had excision followed by 5-FU and betamethasone intralesional injections, group B had 5-FU and betamethasone intralesional injections, and group C had excision followed by radiotherapy. Efficacy parameters were assessed from 8 to 12 months, including improvement on the Vancouver Scar Scale (VSS) and the Patient and Observer Scar Scale (POSAS), as well as side effects and recurrence. Trial registration number: ChiCTR2100046025. Results: After 4 months' treatment, the improvement of the VSS and POSAS scores in group A was not different from that in group C (P > 0.05) but was superior to that in group B (P < 0.05); the pain and pruritus of the three groups were relieved more than 50%. After 8 to 12 months' follow-up, there was no statistical difference in the incidence of side effects and recurrence among the groups (P > 0.05). Conclusion: Excision followed by intralesional low concentrations of 5-FU (12.5mg/mL) with betamethasone is a safe and sustainable treatment for keloid, with no significant difference from excision followed by radiotherapy.

Adrenomedullin Improves Cardiac Remodeling and Function in Obese Rats with Hypertension.

This study aimed to determine whether adrenomedullin (ADM, 7.2 µg/kg/day, ip), an important endogenous active peptide, has a protective role in cardiac remodeling and function in obesity-related hypertension (OH) rats. A high-fat diet (HFD) was used to induce OH for 20 weeks. H9c2 cells incubated with palmitate (PA, 200 µM) to mimic high free fatty acid in obesity were used as an in vitro model. In OH rats, ADM not only decreased body weight (BW) and blood pressure (BP) but also improved systemic inflammation and oxidative stress. Moreover, ADM still had a greater inhibitory effect on local inflammation and oxidative stress in the hearts of OH rats, and the same anti-inflammatory and antioxidant effects were also confirmed in PA-treated H9c2 cells. The ADM receptor antagonist or Akt inhibitor effectively attenuated the inhibitory effects of ADM on inflammation and oxidative stress in PA-stimulated H9c2 cells. Furthermore, ADM application effectively normalized heart function, and hematoxylin-eosin and Masson staining and collagen volume fraction results showed that ADM improved cardiac remodeling in hearts of OH rats. ADM attenuated cardiac inflammation and oxidative stress via the receptor-Akt pathway, which involves the improvement of cardiac remodeling and function in OH rats.

Adrenomedullin ameliorates palmitic acid-induced insulin resistance through PI3K/Akt pathway in adipocytes.

AIMS: White adipose tissue (WAT) dysfunction has been associated with adipose tissue low-grade inflammation and oxidative stress leading to insulin resistance (IR). Adrenomedullin (ADM), an endogenous active peptide considered as an adipokine, is associated with adipocytes function. METHODS: We evaluated the protective effects of ADM against IR in 3T3-L1 adipocytes treated by palmitic acid (PA) and in visceral white adipose tissue (vWAT) of obese rats fed with high-fat diet. RESULTS: We found that endogenous protein expressions of ADM and its receptor in PA-treated adipocytes were markedly increased. PA significantly induced impaired insulin signaling by affecting phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) axis and glucose transporter-4 (GLUT-4) levels, whereas ADM pretreatment enhanced insulin signaling PI3K/Akt and GLUT-4 membrane protein levels, decreased pro-inflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß) and IL-6 levels, and improved oxidative stress accompanied with reduced reactive oxygen species (ROS) levels and increased anti-oxidant enzymes manganese superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx1) and catalase (CAT) protein expressions. Furthermore, ADM treatment not only improved IR in obese rats, but also effectively restored insulin signaling, and reduced inflammation and oxidative stress in vWAT of obese rats. CONCLUSIONS: This study demonstrates a prevention potential of ADM against obesity-related metabolic disorders, due to its protective effects against IR, inflammation and oxidative stress in adipocytes.

Hydrogen sulfide prevents arterial medial calcification in rats with diabetic nephropathy.

BACKGROUND: Arterial medial calcification (AMC) is associated with a high incidence of cardiovascular risk in patients with type 2 diabetes and chronic kidney disease. Here, we tested whether hydrogen sulfide (H2S) can prevent AMC in rats with diabetic nephropathy (DN). METHODS: DN was induced by a single injection of streptozotocin and high-fat diet (45% kcal as fat) containing 0.75% adenine in Sprague-Dawley rats for 8 weeks. RESULTS: Rats with DN displayed obvious calcification in aorta, and this was significantly alleviated by Sodium Hydrosulfide (NaHS, a H2S donor, 50 µmol/kg/day for 8 weeks) treatment through decreasing calcium and phosphorus content, ALP activity and calcium deposition in aorta. Interestingly, the main endogenous H2S generating enzyme activity and protein expression of cystathionine-γ-lyase (CSE) were largely reduced in the arterial wall of DN rats. Exogenous NaHS treatment restored CSE activity and its expression, inhibited aortic osteogenic transformation by upregulating phenotypic markers of smooth muscle cells SMα-actin and SM22α, and downregulating core binding factor α-1 (Cbfα-1, a key factor for bone formation), protein expressions in rats with DN when compared to the control group. NaHS administration also significantly reduced Stat3 activation, cathepsin S (CAS) activity and TGF-ß1 protein level, and improved aortic elastin expression. CONCLUSIONS: H2S may have a clinical significance for treating AMC in people with DN by reducing Stat3 activation, CAS activity, TGF-ß1 level and increasing local elastin level.

Adrenomedullin Attenuates Inflammation in White Adipose Tissue of Obese Rats Through Receptor-Mediated PKA Pathway.

OBJECTIVE: Adrenomedullin (ADM) possesses therapeutic potential for inflammatory diseases. Consequently, the effects of ADM on inflammation in visceral white adipose tissue (vWAT) of obese rats or in adipocytes were explored in this study. METHODS: Male rats were fed a high-fat diet for 12 weeks to induce obesity, and obese rats were implanted with osmotic minipumps providing constant infusion of ADM (300 ng/kg per hour) and continued to be fed a high-fat diet for 4 weeks. RESULTS: When compared with the control group, endogenous protein expression of ADM and ADM receptors in vWAT and in lipopolysaccharide (LPS)-treated adipocytes was markedly increased. ADM significantly decreased the protein expression of the inflammatory mediators TNFα, IL-1ß, cyclooxygenase-2, and inducible nitric oxide synthase in vWAT of obese rats and in adipocytes stimulated by LPS. It also inhibited the activation of the inflammatory signaling pathways MAPK and NF-κB induced by LPS in adipocytes. These effects of ADM in adipocytes were inhibited by the administration of ADM receptor antagonist and cAMP-dependent protein kinase (PKA) activation inhibitor. CONCLUSIONS: ADM can inhibit inflammation in WAT in obesity, which may be mediated by the activation of ADM receptors and PKA.

Adrenomedullin 2 attenuates LPS-induced inflammation in microglia cells by receptor-mediated cAMP-PKA pathway.

Inflammation plays a critical role in the development of neurodegenerative diseases. Adrenomedullin 2 (AM2), a member of the calcitonin gene-related peptide family, has been known to have anti-inflammatory effects. Here, we evaluated the anti-inflammatory effects of AM2 in LPS-activated microglia and BV2 cells. The endogenous mRNA and protein expressions of AM2, calcitonin receptor-like receptor (CLR), receptor activity-modifying proteins (RAMPs) including RAMP1, RAMP2 and RAMP3 and the production of inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected by RT-PCR and Western blot. Our results revealed that LPS (1 µg/mL) significantly stimulated CLR, RAMP1, RAMP2 and RAMP3 protein expressions in BV2 microglia cells, but AM2 had a significant decrease. However, the mRNA levels of AM2, CLR, and RAMP1/2/3 were all markedly increased. LPS also induced obvious increases in mRNA and protein levels of the inflammatory mediators (TNF-α, IL-1ß, COX2 and iNOS). More importantly, AM2 (10 nM) administration effectively inhibited the mRNA and protein expressions of these mediators induced by LPS and increased the cAMP content in LPS-stimulated BV2 cells. Furthermore, the antagonism with AM2 receptor antagonist IMD17-47, adrenomedullin (AM) receptor antagonist by AM22-52 or the inhibition of protein kinase A (PKA) activation by P1195 effectively prevented the inhibitory role of AM2 in LPS-induced production of the above inflammatory mediators. In conclusion, AM2 inhibits LPS-induced inflammation in BV2 microglia cells that may be mainly through AM receptor-mediated cAMP-PKA pathway. Our results indicate AM2 plays an important protective role in microglia inflammation, suggesting therapeutic potential for AM2 in neuroinflammation diseases caused by activated microglia.

Adipose afferent reflex is enhanced by TNFα in paraventricular nucleus through NADPH oxidase-dependent ROS generation in obesity-related hypertensive rats.

BACKGROUND: The adipose afferent reflex (AAR), a sympatho-excitatory reflex, can promote the elevation of sympathetic nerve activity (SNA) and blood pressure (BP). Inflammation in the paraventricular nucleus (PVN) involves sympathetic abnormality in some cardiovascular diseases such as hypertension. This study was designed to explore the effects of tumor necrosis factor alpha (TNFα) in the PVN on the AAR and SNA in rats with obesity-related hypertension (OH) induced by a high-fat diet for 12 weeks. METHODS: Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded in anesthetized rats, and their responses to capsaicin (CAP) stimulation of the right inguinal white adipose tissue were used to evaluate the AAR. RESULTS: Compared to the control rats, the systolic blood pressure (SBP), plasma norepinephrine (NE, indicating SNA) and TNFα levels, TNFα mRNA and protein levels, reactive oxygen species (ROS) content and NADPH oxidase activity in the PVN were significantly elevated in rats with OH. TNFα in the PVN markedly enhanced sympathoexcitation and AAR. Moreover, the enhancement of AAR caused by TNFα can be significantly strengthened by the pretreatment of diethyldithiocarbamate (DETC), a superoxide dismutase inhibitor, but attenuated by TNF-α receptor antagonist R-7050, superoxide scavenger PEG-SOD and NADPH oxidase inhibitor apocynin (Apo) in rats with OH. Acute microinjection of TNF-α into the PVN significantly increased the activity of NADPH oxidase and ROS levels in rats with OH, which were effectively blocked by R-7050. Furthermore, our results also showed that the increased levels of ROS, TNFα and NADPH oxidase subunits mRNA and protein in the PVN of rats with OH were significantly reversed by pentoxifylline (PTX, 30 mg/kg daily ip; in 10% ethanol) application, a cytokine blocker, for a period of 5 weeks. PTX administration also significantly decreased SBP, AAR and plasma NE levels in rats with OH. CONCLUSIONS: TNFα in the PVN modulates AAR and contributes to sympathoexcitation in OH possibly through NADPH oxidase-dependent ROS generation. TNFα blockade attenuates AAR and sympathoexcitation that unveils TNFα in the PVN may be a possible therapeutic target for the intervention of OH.

Intermedin in Paraventricular Nucleus Attenuates Sympathoexcitation and Decreases TLR4-Mediated Sympathetic Activation via Adrenomedullin Receptors in Rats with Obesity-Related Hypertension.

Intermedin/adrenomedullin-2 (IMD/AM2), a member of the calcitonin gene-related peptide/AM family, plays an important role in protecting the cardiovascular system. However, its role in the enhanced sympathoexcitation in obesity-related hypertension is unknown. In this study, we investigated the effects of IMD in the paraventricular nucleus (PVN) of the hypothalamus on sympathetic nerve activity (SNA), and lipopolysaccharide (LPS)-induced sympathetic activation in obesity-related hypertensive (OH) rats induced by a high-fat diet for 12 weeks. Acute experiments were performed under anesthesia. The dynamic alterations of sympathetic outflow were evaluated as changes in renal SNA and mean arterial pressure (MAP) in response to specific drugs. Male rats were fed a control diet (12% kcal as fat) or a high-fat diet (42% kcal as fat) for 12 weeks to induce OH. The results showed that IMD protein in the PVN was downregulated, but Toll-like receptor 4 (TLR4) and plasma norepinephrine (NE, indicating sympathetic hyperactivity) levels, and systolic blood pressure were increased in OH rats. LPS (0.5 µg/50 nL)-induced enhancement of renal SNA and MAP was greater in OH rats than in obese or control rats. Bilateral PVN microinjection of IMD (50 pmol) caused greater decreases in renal SNA and MAP in OH rats than in control rats, and inhibited LPS-induced sympathetic activation, and these were effectively prevented in OH rats by pretreatment with the AM receptor antagonist AM22-52. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor U0126 in the PVN partially reversed the LPS-induced enhancement of SNA. However, IMD in the PVN decreased the LPS-induced ERK activation, which was also effectively prevented by AM22-52. Chronic IMD administration resulted in significant reductions in the plasma NE level and blood pressure in OH rats. Moreover, IMD lowered the TLR4 protein expression and ERK activation in the PVN, and decreased the LPS-induced sympathetic overactivity. These results indicate that IMD in the PVN attenuates SNA and hypertension, and decreases the ERK activation implicated in the LPS-induced enhancement of SNA in OH rats, and this is mediated by AM receptors.

Intermedin in Paraventricular Nucleus Attenuates Sympathoexcitation and Decreases TLR4-Mediated Sympathetic Activation via Adrenomedullin Receptors in Rats with Obesity-Related Hypertension / 神经科学通报·英文版

Intermedin/adrenomedullin-2 (IMD/AM2), a member of the calcitonin gene-related peptide/AM family, plays an important role in protecting the cardiovascular system. However, its role in the enhanced sympathoexcitation in obesity-related hypertension is unknown. In this study, we investigated the effects of IMD in the paraventricular nucleus (PVN) of the hypothalamus on sympathetic nerve activity (SNA), and lipopolysaccharide (LPS)-induced sympathetic activation in obesity-related hypertensive (OH) rats induced by a high-fat diet for 12 weeks. Acute experiments were performed under anesthesia. The dynamic alterations of sympathetic outflow were evaluated as changes in renal SNA and mean arterial pressure (MAP) in response to specific drugs. Male rats were fed a control diet (12% kcal as fat) or a high-fat diet (42% kcal as fat) for 12 weeks to induce OH. The results showed that IMD protein in the PVN was downregulated, but Toll-like receptor 4 (TLR4) and plasma norepinephrine (NE, indicating sympathetic hyperactivity) levels, and systolic blood pressure were increased in OH rats. LPS (0.5 µg/50 nL)-induced enhancement of renal SNA and MAP was greater in OH rats than in obese or control rats. Bilateral PVN microinjection of IMD (50 pmol) caused greater decreases in renal SNA and MAP in OH rats than in control rats, and inhibited LPS-induced sympathetic activation, and these were effectively prevented in OH rats by pretreatment with the AM receptor antagonist AM22-52. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor U0126 in the PVN partially reversed the LPS-induced enhancement of SNA. However, IMD in the PVN decreased the LPS-induced ERK activation, which was also effectively prevented by AM22-52. Chronic IMD administration resulted in significant reductions in the plasma NE level and blood pressure in OH rats. Moreover, IMD lowered the TLR4 protein expression and ERK activation in the PVN, and decreased the LPS-induced sympathetic overactivity. These results indicate that IMD in the PVN attenuates SNA and hypertension, and decreases the ERK activation implicated in the LPS-induced enhancement of SNA in OH rats, and this is mediated by AM receptors.

A subchronic toxicity study, preceded by an in utero exposure phase, with refined arachidonic acid-rich oil (RAO) derived from Mortierella alpina XM027 in rats.

To evaluate the potential toxicity of refined arachidonic acid-rich oil (RAO) derived from Mortierella alpina (M. alpina) XM027, we performed a 90-day subchronic study in F1 Sprague Dawley (SD) rats. This study was preceded by a 4-week pretreatment period of parental (F0) rats and exposure of the F0 dams throughout mating, gestation, and lactation. The results indicated that RAO, at dose levels of 0.5%, 1.5%, and 5%, did not affect either reproductive performance of the parental rats, or any characteristics of the pups. In the subchronic study with the offspring (F1) rats, no treatment related abnormalities were observed. In summary, no observable adverse effect level (NOAEL) in this study was placed at 5% RAO, the highest level tested. This level corresponds to approximately 3750mg/kg in F0 females, 2850mg/kg in F0 males, 4850mg/kg in F1 females, and 4480mg/kg in F1 males.