Targeting RXFP1 by Ligustilide: A novel therapeutic approach for alcoholic hepatic steatosis.

BACKGROUND: Ligustilide (Lig) is the main active ingredient of Umbelliferae Angelicae Sinensis Radix (Chinese Angelica) and Chuanxiong Rhizoma (Sichuan lovase rhizome). Lig possesses various pharmacological properties and could treat obesity by regulating energy metabolism. However, the impact and regulatory mechanism of Lig on alcoholic hepatic steatosis remains unclear. PURPOSE: This study aimed to explore the therapeutic effect of Lig on alcoholic hepatic steatosis and its related pharmacological mechanism. RESULTS: With chronic and binge ethanol feeding, liver tissue damage and lipid accumulation in mice suffering alcoholic hepatic steatosis were significantly improved after Lig treatment. Lig effectively regulated the expression levels of lipid metabolism-related proteins in alcoholic hepatic steatosis. In addition, Lig reduced RXFP1 expression, inhibited the activation of NLRP3 inflammasome, and blocked NET formation. Lig reduced the infiltration of immune cells to the liver and the further prevented the occurrence of alcohol-stimulated inflammatory response in liver. Lig significantly regulated lipid accumulation in alcohol exposed AML12 cells via modulating PPARα and SREBP1. In MPMs, Lig decreased the expression of RXFP1, inhibited the activation of NLRP3 in macrophages stimulated by LPS/ATP, and slowed down the occurrence of inflammatory response. CONCLUSION: Lig sustained lipid metabolism homeostasis in alcoholic hepatic steatosis, through inhibiting the activation of NLRP3 inflammasomes and the formation of NETs, especially targeting RXFP1 in macrophages.

Carnosic acid suppressed the formation of NETs in alcoholic hepatosteatosis based on P2X7R-NLRP3 axis.

BACKGROUND: Alcoholic liver disease (ALD) is accompanied by a disruption of lipid metabolism and an inflammatory response in the liver during the process of disease. Carnosic acid (CA), a natural diterpene extracted from Rosmarinus officinalis (rosemary) and Salvia officinalis (sage), has more pharmacological activities, which is known to be useful in the treatment of obesity and acts by regulating energy metabolism. However, the role and regulation mechanism of CA against ALD remain unclear. HYPOTHESIS: We hypothesized that CA might improve alcoholic-induced hepatosteatosis. STUDY DESIGN AND METHODS: The alcoholic liver disease model was established a mouse chronic ethanol feeding by Lieber-DeCarli control liquid feed (10 d) plus a single binge with or without CA administration. AML12 cells were exposed to ethanol for 24 h. Murine peritoneal macrophages (MPM) were stimulated with LPS and ATP. RESULTS: CA ameliorated lipid accumulation in the liver of mice in the NIAAA model, acting by inhibiting the expression of genes related to lipid synthesis. CA reduced alcohol-induced immune cell infiltration in the liver, and inhibited the activation of P2X7R-NLRP3 inflammasome, meanwhile blocked the formation of NETs in mouse livers tissue. In AML12 cells, CA attenuated the lipid accumulation triggered by ethanol stimulation, which was achieved by inhibiting the expression of SREBP1 and CA reduced the release of inflammatory factor IL-1ß by inhibiting the activation of P2X7R-NLRP3. In MPM, IL-1ß and HMGB1 were reduced after LPS/ATP stimulation in CA-treated cells and supernatant. CONCLUSIONS: CA attenuated alcohol-induced fat accumulation, suppressed the formation of NETs based on P2X7R-NLRP3 axis in mouse livers. Our data indicated that CA exerted hepatoprotective effects, which might be a promising candidate.

Modulation of interleukin-36 based inflammatory feedback loop through the hepatocyte-derived IL-36R-P2X7R axis improves steatosis in alcoholic steatohepatitis.

BACKGROUND AND PURPOSE: Interleukin-36 is induced by proinflammatory cytokines and promotes inflammatory responses, creating an IL-36-based inflammation loop. Although hepatocytes, produce IL-36 responses to drug-induced liver injury, little is known about the mechanistic role of IL-36 signalling during the progression of alcoholic steatohepatitis (ASH). Regarding IL-36/IL-36R and P2X7R coregulating the inflammatory response, we elucidated that modulation of IL-36R-P2X7R-TLR axis affected hepatocyte steatosis as well as the IL-36-based inflammatory feedback loop that accompanies the onset of ASH. EXPERIMENTAL APPROACH: C57BL/6J mice were subjected to either chronic-plus-binge ethanol feeding or acute gavage with multiple doses of ethanol to establish ASH, followed by pharmacological inhibition or genetic silencing of IL-36R and P2X7R. AML12 cells or mouse primary hepatocytes were stimulated with alcohol, LPS plus ATP or Poly(I:C) plus ATP, followed by silencing of IL-36γ, IL-36R or P2X7R. KEY RESULTS: P2X7R and IL-36R deficiency blocked the inflammatory loop, specifically initiated by IL-36 cytokines, in hepatocytes of mice suffering from ASH. Pharmacological inhibition to P2X7R or IL-36R alleviated lipid accumulation and inflammatory response in ASH. IL-36R was indispensable for P2X7R modulated NLRP3 inflammasome activation in ASH, and IL-36 led to a vicious cycle of P2X7R-driven inflammation in alcohol-treated hepatocytes. TLR ligands promoted IL-36γ production in hepatocytes, based on synergism with P2X7R. CONCLUSIONS AND IMPLICATIONS: Blockade of IL-36 based inflammatory feedback loop, via IL-36R-P2X7R-TLRs-modulated NLRP3 inflammasome activation, circumvented steatosis and inflammation that accompanies the onset of ASH, suggesting that targeting IL-36 can serve as a novel therapeutic approach to combat ASH.

Inhibition of HMGB1/TLR4 Signaling Pathway by Digitoflavone: A Potential Therapeutic Role in Alcohol-Associated Liver Disease.

Digitoflavone (DG) is a natural flavonoid abundant in many fruits, vegetables, and medicinal plants. We investigated whether DG inhibits lipid accumulation and inflammatory responses in alcoholic liver disease (ALD) in vivo and in vitro. The mouse ALD model was established by chronically feeding male C57BL/6 mice an ethanol-containing Lieber-DeCarli liquid diet. In vitro, mouse peritoneal macrophages (MPMs) and mouse bone marrow-derived macrophages (BMDMs) were stimulated with LPS/ATP, whereas HepG2 cells and mouse primary hepatocytes were treated with ethanol. DG reduced the serum levels of transaminase and serum and hepatic levels of triglycerides and malondialdehyde in ALD mice. DG downregulated SREBP1 and its target genes and upregulated PPARα and its target genes in the liver of mice with ALD. DG inhibited TLR4-mediated NLRP3 inflammasome activation, consequently reversing the inflammatory response, including the production of HMGB1, IL-1ß, and IL-36γ, as well as the infiltration of macrophages and neutrophils. DG blocked NLRP3/ASC/caspase-1 inflammasome activation and HMGB1 release in LPS/ATP-stimulated MPMs. When Tlr4 was knocked in LPS/ATP-stimulated BMDMs, HMGB1 production and release were blocked, and NLRP3-mediated cleavage and release of IL-1ß was suppressed in Hmgb1-silenced BMDMs. DG amplified these inhibitory effects in Tlr4 or Hmgb1 knockdown BMDMs. In ethanol-exposed hepatocytes, DG reduced lipogenesis and promoted lipid oxidation by inhibiting the HMGB1-TLR4 signaling pathway while suppressing the inflammatory response induced by ethanol exposure. Our data demonstrated that DG inhibited the occurrence of lipid accumulation and the inflammatory response via the HMGB1-TLR4 axis, underscoring a promising approach and utility of DG for the treatment of ALD.

[Endovascular management of symptomatic carotid stenosis combined with kinking].

OBJECTIVE: To study the necessity, feasibility, security of carotid angioplasty and stenting (CAS) for symptomatic carotid stenosis combined with kinking. METHODS: Twelve patients with symptomatic carotid stenosis and kinking demonstrated by digital subtraction angiography (DSA) received CAS from December 2003 to December 2009. There were 9 male and 3 female patients, age ranged from 59 to 77 years (mean 69.3 years). All the patients' clinical, imaging, intervention and follow up data were collected and analyzed. RESULTS: All CAS procedures were successfully performed with 14 self-expandable stents placed. The mean degree of stenosis was reduced from 85.6% before stenting to 11.2% after stenting, the angle of kinking, according to Metz' category, were improved from less than 90° to more than 120° in each case. No perioperative procedure related stroke and transient ischemic attack (TIA) occurred. The clinical symptoms and signs of cerebral ischemia were improved or disappeared for all patients. During follow-up of these 12 patients for 6 to 72 months, one patient experienced ipsilateral carotid territory TIA and another patient experienced contralateral carotid territory TIA. DSA follow up of 5 patients demonstrated 1 case with in-stent restenosis and arterial kinking remote to the stent of internal carotid artery. CAS were performed again and CT angiography follow up demonstrated no kinking and restenosis 2 years after the intervention. Duplex scan of the other 7 patients demonstrated neither kinking nor restenosis. CONCLUSIONS: CAS seems to be feasible and safe for the patients with symptomatic kinking and stenosis, and maybe helpful to lower the risk of cerebral ischemia, but further study is needed.

[Endovascular treatment of micro-intracranial aneurysms: adverse factors of micro-catheterization and its relevant managements].

OBJECTIVES: To investigate adverse factors that may hinder successful placement and stabilization of the microcatheter during endovascular therapy of micro-intracranial aneurysms (≤ 3 mm in maximum diameter), and to explore the relevant managements. METHODS: Forty-six patients with fifty-one micro-intracranial aneurysms treated by endovascular therapy from June 2001 to October 2009 were retrospectively analyzed for their intervention data. RESULTS: Adverse factors of optimal micro-catheterization mainly included, tortuosity of the proximal vessels (PVs) and the parent artery (PA), relative large gap in diameter among the PVs, the PA and the microcatheter, relative large divergence in direction among the PVs, the PA and the aneurysm dome, and stent deployed in the PA. CONCLUSIONS: Carefully considering the direction of the PVs and the PA, the aneurysm's location and dome orientation, choosing the microcatheter and microwire after balancing among their physical properties, as well as utilizing balloon and/or stent assistance, can facilitate micro-catheterization during endovascular treatment of micro-intracranial aneurysms.

[Endovascular treatment of ruptured and unruptured intracranial very small aneurysms].

OBJECTIVE: To evaluate the feasibility and safety of endovascular treatment of ruptured and unruptured intracranial very small aneurysms (< or = 3 mm in maximal diameter). METHODS: Forty-eight intracranial very small aneurysms in 44 patients treated with endovascular therapy from June 2001 to August 2009 were reviewed retrospectively in clinical, imaging, interventional and follow-up data. Among 44 patients, there were 20 males and 24 females with a mean age of 57.8 years old. The Hunt-Kosnik grade was as follows: Grade 0 (n = 11); Grades I & II (n = 23); Grades III & IV (n = 9); and ungraded (n = 1). The sizes of 48 aneurysms were not more than 3 mm in maximal diameter. The locations of aneurysms were as follows: ACoA (n = 11), MCA (n = 8), PCoA (n = 14), ICA (n = 12), pericallosal artery (n = 1), VA (n = 1) and PICA (n = 1). Thirty-nine aneurysms were embolized with coil, of which 13 with stent assistance and 6 by balloon remodeling technique. The other 9 aneurysms underwent sole stent placement in parent artery. RESULTS: Among 39 coiling aneurysms, 100% occlusion was achieved in 9 aneurysms, 90% in 20, 80% in 9 and less than 80% in 1 respectively. Only one aneurysm ruptured during coiling. Two patients had transient hemiparesis and one patient had ataxia caused by bilateral cerebellar infarction postoperatively. All patients were clinically followed up for 4-90 months and no recurrent hemorrhage occurred. Thirteen patients received repeat angiography at 4-72 months post-treatment. And no radiological re-growth was detected. CONCLUSION: Endovascular treatment of ruptured and unruptured intracranial very small aneurysms seems to be technically feasible, relatively safe and practically effective.

[Changes of nitric oxide and endothelin serum level after carotid balloon denudation or stent assisted angioplasty: an experimental and clinical observation].

OBJECTIVE: To observe the changes of nitric oxide (NO) and endothelin (ET) serum level in the Guangxi BA-MA minipigs whose carotid arteries were injured by balloon denudation and in the patients with carotid stent assisted angioplasty. METHODS: Twelve Guangxi BA-MA minipigs were chosen. High fat/cholesterol feeding and endovascular balloon denudation were used to create a carotid artery atherosclerotic stenosis animal model. Blood samples were collected from peripheral veins before starting the procedure, and again, at 2 and 3 weeks after the procedure, respectively. Serum NO and ET concentrations of blood samples were tested. Nineteen patients with carotid artery stenosis who underwent stent assisted angioplasty were randomly selected, and their serum NO and ET were tested using the same methods as above. RESULTS: In the animal group, there was a significant decrease of mean NO concentration at 2 weeks after carotid injury (t-test, P < 0.05), however, no significant change of ET was observed. A very significant increase of ET was observed at 3 weeks after the procedure (t-test, P < 0.01). In the patient group, there were no significant differences among serum NO or ET concentration of peripheral vein blood before, immediately after, and 6 h after the endovascular treatment. CONCLUSIONS: In this study, a decrease of NO concentration and an increase of ET concentration of peripheral vein blood are found in BA-MA minipigs after carotid arteries are injured by balloon denudation, which might be a cue for the formation of atherosclerosis. However, no significant changes are observed in this group of patients who underwent carotid angioplasty treatment. Therefore, further studies are needed.

[Evaluation of cerebral hemodynamics in geriatric carotid stenosis pre- and post-carotid artery stenting].

OBJECTIVES: To explore influencing factors of regional cerebral blood flow (rCBF) in geriatric carotid stenosis, and to analyze changes of rCBF and clinical symptoms after carotid stenting. METHODS: During August 2005 and April 2008, 68 geriatric patients of carotid stenosis having SPECT examination in our hospital were retrospectively studied, whose diagnosis was approved by angiography. Correlated rCBF was compared separately in different stenotic degrees of carotid stenosis, in unilateral or bilateral stenosis, accompanied with vertebrobasilar stenosis (VBS) or not, with collateral circulation or not, before and after carotid stenting. RESULTS: When patients of unilateral carotid stenosis were grouped by different clinical factors, cases of patients with reduced rCBF were compared using chi(2) test: the P value was 0.046 and 0.020 when comparing group of stenotic degree 90% - 99% with group 70% - 89% and group 50% - 69%; the P value was 0.927 between group accompanied with VBS and group without; the P value was 0.222 between group with collateral circulation and group without. When comparing reduced rCBF cases between unilateral and bilateral carotid stenosis, the P value was 0.046. After carotid stenting, 76% of patients had their rCBF improved, and also the scores of presenting symptoms evaluated by modified Rankin scale were elevated from 1.4 +/- 0.7 on admission to 0.4 +/- 0.3 postoperatively (P < 0.001). CONCLUSIONS: The research indicates that higher stenotic degree and bilateral carotid stenosis may cause rCBF decrease in geriatric carotid stenosis. Carotid stenting may improve rCBF and change clinical symptoms significantly.

[Clinical study and numerical simulation of hemodynamics in the tortuosity of internal carotid artery].

OBJECTIVES: To establish a theoretical model for the tortuosity of internal carotid artery and summarize the hemodynamic rule of blood flow in a tortuous artery. To explore the relationship of cerebral ischemia and tortuous internal carotid artery. METHODS: Taking the internal carotid artery as a prototype, a geometric model of a tortuous artery was constructed according to the normal physiological and anatomical parameters of internal carotid artery. The boundary conditions and calculation conditions of blood flow are proposed. The numerical simulation of the blood flow in the tortuous artery is carried out with finite element method. Hemodynamic parameters of internal carotid artery were measured in 15 cases with the tortuosity of internal carotid artery and in 15 cases of normal control group. Blood pressure was measured by microcatheter connecting a pressure transducer at internal carotid artery, pre-tortuous and post-tortuous artery. The diameter and length of the above artery were measured and calculated by DSA machine. RESULTS: Numerical simulation results indicated pressure drop of blood flow and elongated length of artery is increased with diminution of the angle of tortuous artery. Clinical measurement data disclosed the same trend in the same curve as numerical simulation. CONCLUSION: The elongation and tortuosity of internal carotid artery results in decrease of blood pressure in the distal segment of tortuous internal carotid artery, kinking of internal carotid artery may be one of factors related to attack of cerebral ischemia on certain conditions.

[Management of elderly patients with symptomatic vertebrobasilar insufficiency].

OBJECTIVE: To evaluate the feasibility, safety and short-term efficacy of stent-assisted angioplasty and/or drug therapy for elderly patients with symptomatic vertebrobasilar insufficiency. METHODS: Elderly patients (> or = 60 years old) with symptomatic vertebrobasilar stenosis (> or = 50%) demonstrated by cerebral angiography were treated with drug therapy and some with endovascular stenting further from April 2001 to June 2006. The clinical, imaging, intervention and follow-up data were collected and analyzed. RESULTS: Eighty-one elderly patients were chosen for study, including 68 males and 13 females. The mean age is 70 years (60 - 87 years); stroke rate of 4.9% (4/81) and stroke-related mortality rate of 2.5% (2/81) were found in this group during hospitalization and follow-up (mean 28.1 months), and symptoms resolved or improved clinically in 66 (81.5%). Fifty-two balloon expandable stents were placed in 48 (59.3%) patients of this group with a technical success rate of 98.1% and the mean degree of stenosis was reduced from (82.4 +/- 13.1)% to (6.4 +/- 3.2)% (t = 22.4, P = 0.00). CONCLUSIONS: Appropriate management including endovascular stenting and/or drug therapy may improve short-term outcomes of elderly patients with symptomatic vertebrobasilar insufficiency; meanwhile, stent-assisted angioplasty is technically feasible and relatively safe.