Mechanisms of Renal-Splenic Axis Involvement in Acute Kidney Injury Mediated by the α7nAChR-NF-κB Signaling Pathway.

This study aimed to investigate the effect of splenectomy on dexmedetomidine-activated cholinergic anti-inflammatory pathway-mediated alleviation of LPS-induced AKI. A mouse model of septic kidney injury was established in C57BL/6 mice. A total of 30 C57BL/6 mice were randomly divided into the control group, LPS group, dexmedetomidine + LPS group, splenectomy group, splenectomy + LPS group, and splenectomy + dexmedetomidine + LPS group. The pathological effects in kidney tissues in each group were analyzed by HE staining. Apoptosis in each group was examined by the TUNEL method. Cr and Cys-C levels in each group were measured by ELISA. The expression levels of IL-6, NF-κB p65, Caspase-3, the antiapoptotic protein Bcl-2, the proapoptotic protein Bax, and α7nAChR in each group were measured by qRT-PCR and Western blotting. Dexmedetomidine alone reduced apoptosis in kidney tissue; however, apoptosis was increased after splenectomy in mice treated with dexmedetomidine. Splenectomy reduced the production of proinflammatory cytokines in circulation and had a protective effect on the kidney. Splenectomy inhibited dexmedetomidine-mediated activation of the α7nAChR pathway. Dexmedetomidine effectively alleviated LPS-induced kidney injury, and splenectomy inhibited the anti-inflammatory, antiapoptotic, and renoprotective effects of dexmedetomidine. The kidney-spleen axis is mediated by the α7nAChR-NF-κB signaling pathway and is involved in the development of AKI.

Effect of splenectomy on attenuation of LPS-induced AKI through GTS-21-induced cholinergic anti-inflammatory pathway.

This work was undertaken to explore the role of splenectomy on attenuation of lipopolysaccharide (LPS)-induced acute kidney injury (AKI) through GTS-21-induced cholinergic anti-inflammatory pathway. C57BL/6 mice were used to construct models of sepsis-induced renal injury. HE, Tunel and blood assays were used to determine the success of the model. The animals were examined after splenectomy with or without LPS and GTS-21+LPS treatments. The pathological changes and apoptosis in the renal tissue were detected using HE and Tunel assays. The contents of creatinine (Cr) and cystatin-C (Cys-C) were measured using ELISA. The expression of IL-6, NF-kB p65, Caspase-3, anti-apoptotic protein Bcl-2, apoptotic protein Bax and α7nAChR was quantified using qRT-PCR. The expression of Bcl-2, Bax, Caspase-3, IL-6, NF-kB p65, α7nAChR and p-STAT3 was using assessed using Western blot analysis. HE, Tunel, BUN and serum creatinine (SC) assay showed that renal injury models were successfully established. Compared with the control, the apoptosis in the LPS group was significantly increased and decreased after GTS-21 treatment. However, splenectomy combined with GTS-21 increased the apoptosis, indicating that splenectomy could partially offset the anti-apoptosis effect of GTS-21. In animals treated with LPS, the contents of Cr and Cys-C increased significantly. These contents reduced following GTS-21 treatment, but increased after splenectomy. After LPS treatment, the expression of IL-6, NF-kB p65, p-STAT3, Caspase-3 and Bax was significantly up-regulated, while the expression of α7nAChR and Bcl-2 significantly down-regulated. Compared with LPS treated mice, splenectomy reduced the expression of IL-6, NF-kB p65 and p-STAT3, suggesting that splenectomy inhibits the activation of α7nAChR pathway by the GTS-21. It is clear that GTS-21 effectively attenuates LPS-induced renal injury; splenectomy suppresses the anti-inflammatory and anti-apoptosis activity and renal protective effect of GTS-21. On other hand, splenectomy reduces the production of inflammatory cytokines in the circulation, and has certain protective effect on the kidney. Therefore, the impact of splenectomy on LPS-induced AKI depends on the strength of the two aspects.

GTS-21 Protected Against LPS-Induced Sepsis Myocardial Injury in Mice Through α7nAChR.

Sepsis-induced myocardial injury is a well-known cause of mortality. The cholinergic anti-inflammatory pathway (CHAIP) is a physiological mechanism by which the central nervous system regulates immune response through the vagus nerve and acetylcholine; the α7-nicotinic acetylcholine receptor (α7nAChR) is the main component of CHAIP; GTS-21, a synthetic α7nAChR selective agonist, has repeatedly shown its powerful anti-inflammatory effect. However, little is known about its effect on LPS-induced myocardial injury. We investigated the protective effects of GTS-21 on lipopolysaccharide (LPS)-induced cardiomyopathy via the cholinergic anti-inflammatory pathway in a mouse sepsis model. We constructed the model of myocardial injury in sepsis mice by C57BL/6 using LPS and determined the time of LPS treatment by hematoxylin-eosin (HE) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). C57BL/6 mice were randomized into five groups: blank control group, model group, α-bungarotoxin + LPS group, GTS-21 + LPS group, and α-bungarotoxin + GTS-21 + LPS group. The pathological results of myocardial tissue were detected by the HE method; the apoptosis rate was detected by the TUNEL method; the relative expressions of NF-κB p65, Caspase-3, Caspase-8, Bcl-2, Bax, p53, and a7nAChR were detected by real-time quantitative PCR (RT-PCR); and the protein expressions of IL-6, IL-1 ß, TNF-α, and pSTAT3 were detected by western blot. The results showed that LPS-induced myocardial pathological and apoptosis changes were significant compared with the blank group, which was reversed by GTS-21; however, pretreatment with α-bungarotoxin obviously blocked the protective effect of GTS-21. NF-κB p65, Caspase-3, Caspase-8, Bax, p53, IL-6, IL-1ß, TNF-α, and pSTAT3 were significantly increased in the model group, while a7nAChR and Bcl-2 were significantly decreased; GTS-21 treatment reversed that result, while pretreatment with α-bungarotoxin strengthened the result in the model. And pretreatment with α-bungarotoxin blocked the protective effect of GTS-21. GTS-21 can alleviate the LPS-induced damage in the heart via a7nAChR, and pretreatment with α-bungarotoxin obviously blocked the protective effect of GTS-21 on sepsis in mice.

Dexmedetomidine protects against lipopolysaccharide-induced sepsis-associated acute kidney injury via an α7 nAChR-dependent pathway.

Acute kidney injury (AKI) is a clinical syndrome that results in severe tubular damage with high morbidity and mortality. However, there is a lack of effective therapy strategies. Therefore, it is critical to develop effective drugs for AKI. Dexmedetomidine (DEX), a highly selective α2-adrenoreceptor agonist, has neuroprotective, anti-inflammatory and sympatholytic properties. The present study aimed to investigate the effect DEX on attenuating the inflammatory reaction and apoptosis in the kidney tissues of septic mice and to explore its underlying mechanisms. Sepsis-induced AKI mice models were generated via intraperitoneal injection of lipopolysaccaride (LPS). DEX reduced LPS-induced local inflammation and tubular apoptosis, which was aggravated in the pathogenesis of renal dysfunction. Reverse transcription-quantitative polymerase chain reaction and western blot analysis results revealed that the expression of pro-apoptotic genes and inflammatory factors were markedly reduced by DEX pretreatment. Furthermore, the protective role of DEX was markedly inhibited by the α7 nicotinic acetylcholine receptor (nAChR) antagonist α-bungarotoxin. These findings provided novel evidence for the anti-apoptotic and anti-inflammatory effects of DEX in LPS-induced AKI mice through an α7 nAChR-dependent signaling pathway.

Dexmedetomidine alleviates LPS-induced septic cardiomyopathy via the cholinergic anti-inflammatory pathway in mice.

This study was conducted to investigate the role of the cholinergic anti-inflammatory pathway in LPS-induced septic cardiomyopathy in mice. C57BL/6 mice were used to construct septic cardiomyopathy models. The optimal duration of lipopolysaccharide (LPS) treatment was determined by HE staining and TUNEL assay. Blank controls were intraperitoneally injected with saline and models were injected with LPS (10 mg/kg) (LPS), α-bungarotoxin (BT-LPS), BT and dexmedetomidine (BT-DEX-LPS). The pathological examinations were performed on HE- stained myocardium tissues, apoptosis was determined using TUNEL assay, mRNA expression of NF-κB p65, Caspase-3, Caspase-8, Bcl-2, Bax, p53 and α7nACh was quantified using qRT-PCR, protein levels of IL-6, IL-1ß, TNF-α and phosphorylated STAT3 (p-STAT3) were analyzed using Western blot analysis. HE staining and TUNEL assays showed that the optimal LPS treatment time for septic cardiomyopathy induction was 16 h. Compared with the blank control, mice in LPS group had significantly higher apoptosis, while DEX and BT reduced apoptosis when they were used separately and increased apoptosis when they were used jointly. In the LPS-treated mice, the levels of NF-κb p65, Caspase-3, Caspase-8, Bax, p53, IL-6, IL-1ß, TNF-α and p-STAT3 were significantly increased, while α7nAChR level was decreased significantly (P < 0.01); DEX alone had no impact on the expression of these proteins but significantly up-regulated the expression of these genes except α7nAChR when used jointly with BT (P < 0.01). It is clear that DEX can alleviate heart injury, while α7nAChR-specific blocker BT is antagonistic against the anti-inflammatory effect of DEX on sepsis in mice.

GTS-21 attenuates LPS-induced renal injury via the cholinergic anti-inflammatory pathway in mice.

This study aimed to investigate the role of GTS-21 in cholinergic anti-inflammatory pathway-mediated protection of LPS-induced septic renal injury in mice. C57BL/6 mice were used to construct septic injury models. The optimal duration of lipopolysaccharide (LPS) treatment was determined using HE staining and TUNEL assay. Mice injected with saline were used as blank control and with LPS (10 mg/kg) as model, which were further treated with α-bungarotoxin (BT-LPS), GTS-21 (GTS-21-LPS) and BT and GTS-21 (BT-GTS-21-LPS). The pathological examinations were performed on HE stained renal tissues, apoptosis was determined using TUNEL assay, mRNA expression of NF-kB p65, Caspase-3, Caspase-8, Bcl-2, Bax, p53 and a7nACh was quantified using qRT-PCR, protein levels of IL-6, IL-1ß, TNF-α and phosphorylated STAT3 (p-STAT3) were analyzed using Western blots. HE staining and TUNEL assays showed that the optimal LPS treatment time for renal injury induction was 16 h. Compared with the blank control, mice in LPS group had significantly higher levels of NF-Kb p65, Caspase-3, Caspase-8, Bax, p53, IL-6, IL-1ß, TNF-α and p-STAT3, while α7nAChR and Bcl-2 levels were decreased significantly (P < 0.01); GTS-21 and BT significantly increased the expression of NF-Kb p65, Caspase-3, Caspase-8, Bax, p53, IL-6, IL-1ß, TNF-α and p-STAT3, while α7nAChR and Bcl-2 levels were decreased significantly (P < 0.01). It is concluded that GTS-21 can effective alleviate the renal injury, while α7nAChR-specific blocker BT is antagonistic against the anti-inflammatory effect of GTS-21 on sepsis in mice.

The calcium-sensing receptor R990G polymorphism is associated with increased risk of hypertriglyceridemia in obese Chinese.

BACKGROUND: We have demonstrated that the calcium-sensing receptor (CaSR) is involved in lipid metabolism; however, whether CaSR polymorphisms affect lipid metabolism in obesity is still unclear. The present study aimed to determine the effects of CaSR polymorphisms on HTG risk in obese Chinese. METHODS: A total of 972 subjects with HTG and 1197 with normal triglyceride (NTG) were stratified by body mass index (BMI) into normal weight, overweight or obesity subgroups. After 12-h fasting, CaSR polymorphisms in exon 7 were determined in the blood. Serum lipids and glucose, as well as height, body weight and waist circumference were measured. The anthropometric and metabolic characteristics of the NTG subjects were re-evaluated 3 years later. RESULTS: There were no genotypic or allelic distribution differences for the A986S or Q1011E polymorphisms between the NTG and HTG groups. However, the G/G genotypic and G allelic distributions of the CaSR R990G polymorphism in the HTG group were higher than the NTG group (p<0.001). After stratification, in obese subjects, the homozygous (G/G) distribution of the CaSR R990G polymorphism in the HTG group was significantly higher than in the NTG group (p=0.001), and showed an increased risk of HTG at baseline [OR=2.55, 95% CI=1.65-3.92, p<0.006]. Interaction of the CaSR R990G polymorphism with BMI was associated with increased risk of HTG (ß=0.927, p<0.001). Re-evaluation of the NTG subjects revealed significantly increased serum triglyceride levels in obese homozygous versus wildtype carriers (p<0.05). CONCLUSIONS: These results suggest that the CaSR R990G polymorphism is associated with increased risk of HTG, especially in obese Chinese, and may be a potential genetic predictor of diseases related to HTG.