Betulinic acid (BA), a pentacyclic triterpenoid, has been reported to inhibit cardiovascular dysfunction under sepsis-induced oxidative stress. Nuclear factor erythroid-2 related factor-2 (Nrf2) is regarded as a key transcription factor regulating expression of endogenous antioxidative genes. To explore the preventive effects of BA against vascular hyporeactivity and the related antioxidative mechanism in sepsis, contraction and relaxation in aortas isolated from lipopolysaccharide (LPS)-challenged rats were performed. Male Sprague-Dawley rats were pretreated with brusatol (Bru, 0.4 mg/kg/2 days, i.p.), an inhibitor of Nrf2, and BA (10, 25, 50 mg/kg/day, i.g.) for 3 days and injected with LPS (10 mg/kg, i.p.) at the 4th day. Rats were anesthetized and killed by cervical dislocation after they were treated with LPS for 4 h. Thoracic aortas were immediately dissected out to determine contraction and relaxation using the organ bath system. Pro-inflammatory factors interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and oxidative stress were measured in aortic tissues and plasma. mRNA expression of Nrf2-regulated antioxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and heme oxygenase-1 (HO-1), in rat aortas was determined. Increases of IL-1β, TNF-α, nitric oxide, and malondialdehyde and the decrease of glutathione induced by LPS were significantly attenuated by pretreatment with different doses of BA in plasma and aortas (p < 0.05 versus LPS), all of which were blocked by Bru (p < 0.01). Inhibition of phenylephrine (PE)- and KCl-induced contractions and acetylcholine (ACh)-induced vasodilatation in aortas from LPS-challenged rats was dose-dependently reduced by BA (p < 0.05; percentage improvements by BA in PE-induced contraction were 55.38%, 96.41%, and 104.33%; those in KCl-induced contraction were 15.11%, 23.96%, and 22.96%; and those in ACh-induced vasodilatation were 16.08%, 42.99%, and 47.97%), all of which were reversed by Bru (p < 0.01). Improvements of SOD, GPx, and HO-1 mRNA expression conferred by BA in LPS-challenged rat aortas were inhibited by Bru (p < 0.01; 145.45% versus 17.42%, 160.69% versus 22.76%, and 166.88% versus 23.57%). These findings suggest that BA attenuates impairments of aortic contraction and relaxation in LPS-challenged rats by activating Nrf2-regulated antioxidative pathways.

The contribution of the cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) and adrenomedullin (ADM) to the developmental modulation of stressful events remains controversial. This study explored the effects of endogenous ADM in the CSF-contacting nucleus on immobilization of stress-induced physiological parameter disorders and glucocorticoid hormone releasing hormone (CRH), rat plasma corticosterone expression, and verification of such effects by artificially lowering ADM expression in the CSF-contacting nucleus by targeted ablation of the nucleus. Immunohistochemical experiments showed that ADM-like immunoreactivity and the calcitonin receptor-like receptor (CRLR) marker were localized in the CSF-contacting nucleus. After 7 continuous days of chronic immobilization stress (CIS), animals exhibited anxiety-like behavior. Also, an increase in serum corticosterone, and enhanced expression of ADM in the CSF-contacting nucleus were observed, following activation by CIS. The intracerebroventricular (i.c.v.) administration of the ADM receptor antagonist AM22-52 significantly reduced ADM in the CSF-contacting nucleus, additionally, blocked the effects of ADM, meaning the expression of CRH in the hypothalamic paraventricular nucleus (Pa) and serum corticosterone level were increased, and the physiological parameters of the rats became correspondingly deteriorated. Additionally, the i.c.v. administration of cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to a cholera toxin subunit, completely eliminated the CSF-contacting nucleus, worsening the reaction of the body to CIS. The collective results demonstrated that ADM acted as a stress-related peptide in the CSF-contacting nucleus, and its lower expression and blocked effects in the nucleus contributed to the deterioration of stress-induced physiologic parameter disorders as well as the excessive expressions of stress-related hormones which were part of the hypothalamic-pituitary-adrenal (HPA) axis.

This study was undertaken to determine AM expression in carbon tetrachloride (CCl4)-induced liver cirrhosis developed with peritoneal ascites. Sprague-Dawley rats received subcutaneous injections of CCl4 twice weekly in olive oil (1:1, 0.3 ml per kg body weight) for 6 or 12 weeks until ascites developed, or saline in olive oil as control. At 6 weeks, fibrosis developed and at 12 weeks cirrhosis developed with ascites formation. At both 6 and 12 weeks, increases in plasma renin and AM were evident, as was the gene expression of AM. At 12 weeks after CCl4 injection, the gene expression of calcitonin-like-receptor (CRLR) and receptor activity modifying proteins (RAMP1, RAMP2 and RAMP3) were all elevated when compared to the control. The results suggest that liver cirrhosis increases mRNA expressions of AM, CRLR and RAMP1, RAMP2 and RAMP3 and that the increase in AM gene expression precedes the development of cirrhosis. The increase in AM synthesis as reflected by an increase in AM gene expression, together with a lack of increase in AM peptide at both 6 and 12 weeks may suggest an elevation of AM release. Given the potent vasodilatory action of AM, the increase in the synthesis and release of AM in the cirrhotic liver may also contribute to peripheral vasodilatation in liver cirrhosis.

Adrenomedullin (AM) is a 52 amino acid peptide that plays a critical role in several diseases such as hypertension, cancer, diabetes, cardiovascular and renal disorders, among others. Interestingly, AM behaves as a protective agent against some pathologies, yet is a stimulating factor for other disorders. Thus, AM can be considered as a new and promising target for the design of non-peptidic modulators that could be useful for the treatment of those pathologies, by regulating AM levels or the activity of AM. A full decade on from its discovery, much more is known about AM molecular biology and pharmacology, but this knowledge still needs to be applied to the development of clinically useful drugs.

The aim of the present study was to determine the temporal changes in tissue adrenomedullin (AM) and cytokine contents and cytokine and preproAM mRNA levels in the kidney, liver, adrenal gland and spleen of lipopolysaccharide (LPS)-treated rats. Rats were injected with LPS (10 mg/kg, i.p.). Radioimmunoassay and solution hybridization-RNase protection assays were used to follow the changes in AM and its mRNA levels, respectively; ELISA and reverse transcription-polymerase chain reaction were used to follow the changes in cytokines and their mRNA levels, respectively. In the kidney, the preproAM mRNA levels were increased 1 and 3 h after LPS treatment, whereas AM levels were decreased at 3 h. Interleukin (IL)-6 and IL-1beta levels were increased at 3 and 6 h, respectively. The preproAM mRNA levels were elevated in the liver 3 h after LPS injection. Concentrations of tumour necrosis factor (TNF)-alpha and IL-1beta were increased at l and 6 h, respectively. There were no changes in the levels of either preproAM mRNA or AM in the adrenal gland and the spleen. In the spleen, TNF-alpha levels were elevated at 1 and 3 h after LPS injection and IL-1beta was elevated at 1 and 6 h after LPS injection, whereas in the adrenal gland IL-1beta was elevated at 6 h after injection. The mRNA levels of the three cytokines were elevated at all three time intervals examined in the kidney, liver, adrenal gland and spleen, with the exception that TNF-alpha mRNA was not elevated in the adrenal gland at 6 h after LPS injection and IL-1beta mRNA was not elevated in the spleen at 3 and 6 h. The plasma concentrations of TNF-alpha were increased at 1 and 3 h after LPS injection, whereas plasma concentration of IL-1beta and IL-6 were elevated at 3 and 6 h for both. The present results suggest that the biosynthesis and secretion of AM may be differentially regulated in various tissues of rats injected with LPS and that AM may interact with cytokines during inflammation.