BACKGROUND: To observe the effects of Chinese medicine (CM) Polygonum cuspidatum (PC) on adenosine 5'-monophosphate-activated protein kinase (AMPK), forkhead box O3α (FOXO3α), Toll-like receptor-4 (TLR4), NACHT, LRR and PYD domains-containing protein 3 (NLRP3), and monocyte chemoattractant protein-1 (MCP-1) expression in a rat model of uric acid-induced renal damage and to determine the molecular mechanism. METHODS: A rat model of uric acid-induced renal damage was established, and rats were randomly divided into a model group, a positive drug group, and high-, medium-, and low-dose PC groups (n=12 per group). A normal group (n=6) was used as the control. Rats in the normal and model groups were administered distilled water (10 mL•kg) by intragastric infusion. Rats in the positive drug group and the high-, medium-, and low-dose PC groups were administered allopurinol (23.33 mg•kg), and 7.46, 3.73, or 1.87 g•kg•d PC by intragastric infusion, respectively for 6 to 8 weeks. After the intervention, reverse transcription polymerase chain reaction, Western blot, enzyme linked immunosorbent assay, and immunohistochemistry were used to detect AMPK, FOXO3α, TLR4, NLRP3, and MCP-1 mRNA and protein levels in renal tissue or serum. RESULTS: Compared with the normal group, the mRNA transcription levels of AMPK and FOXO3α in the model group were significantly down-regulated, and protein levels of AMPKα1, pAMPKα1 and FOXO3α were significantly down-regulated at the 6th and 8th weeks (P<0.01 or P<0.05). The mRNA transcription and protein levels of TLR4, NLRP3 and MCP-1 were significantly up-regulated (P<0.01 or P<0.05). Compared with the model group, at the 6th week, the mRNA transcription levels of AMPK in the high- and medium-dose groups, and protein expression levels of AMPKα1, pAMPKα1 and FOXO3α in the high-dose PC group, AMPKα1 and pAMPKα1 in the mediumdose PC group, and pAMPKα1 in the low-dose PC group were significantly up-regulated (P<0.01 or P<0.05); the mRNA transcription and protein levels of TLR4 and NLRP3 in the 3 CM groups, and protein expression levels of MCP-1 in the medium- and low-dose PC groups were down-regulated (P<0.01 or P<0.05). At the 8th week, the mRNA transcription levels of AMPK in the high-dose PC group and FOXO3α in the medium-dose PC group, and protein levels of AMPKα1, pAMPKα1 and FOXO3α in the 3 CM groups were significantly up-regulated (P<0.01 or P<0.05); the mRNA transcription levels of TLR4 in the medium- and low-dose PC groups, NLRP3 in the high- and low-dose PC groups and MCP-1 in the medium- and low-dose PC groups, and protein expression levels of TLR4, NLRP3 and MCP-1 in the 3 CM groups were down-regulated (P<0.01 or P<0.05). CONCLUSION PC up-regulated the expression of AMPK and its downstream molecule FOXO3α and inhibited the biological activity of TLR4, NLRP3, and MCP-1, key signal molecules in the immunoinflammatory network pathway, which may be the molecular mechanism of PC to improve hyperuricemia-mediated immunoinflflammatory metabolic renal damage.
AMP-Activated Protein Kinases ; physiology ; Animals ; Chemokine CCL2 ; blood ; Disease Models, Animal ; Fallopia japonica ; Forkhead Box Protein O3 ; physiology ; Hyperuricemia ; complications ; Kidney Diseases ; drug therapy ; etiology ; Male ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Uric Acid

OBJECTIVETo explore the mechanisms of neuroprotective effects of c-Jun N-terminal kinase (JNK)/FOXO3a transcription factor signaling pathway inhibition on hypoxic-ischemic neuronal apoptosis in neonatal rats with hypoxic-ischemic brain damage (HIBD).

METHODSSixty-four 7-day-old Sprague-Dawley rats were divided into four groups: hypoxia-ischemia (HI), sham-operated, JNK specific inhibitor AS601245-treated, and DMSO vehicle. Rats' cerebral cortexes were collected at 24 hours after HI. Western blot was used to detect the protein expression of JNK, p-JNK, FOXO3a, nuclear and cytoplasmic FOXO3a, Bim, and CC3. TUNEL staining was used to detect the apoptotic cells.

RESULTSCompared with the sham-operated group, p-JNK protein increased (P<0.01), nuclear protein of FOXO3a increased (P<0.01), cytoplasmic protein decreased (P<0.01), and pro-apoptotic proteins Bim and CC3 increased 24 hours after HI (P<0.01). Compared with the HI and DMSO vehicle groups, p-JNK protein was reduced (P<0.01), nuclear protein of FOXO3a was also reduced (P<0.01), cytoplasmic protein increased (P<0.01), and Bim and CC3 proteins decreased (P<0.01) in the AS601245-treated group 24 hours after HI. TUNEL positive cells were reduced in the AS601245-treated rats compared with the HI and DMSO vehicle groups 24 hours after HI (P<0.01).

CONCLUSIONSJNK activity increases in the neonatal rat brain with HI damage. JNK activity inhibition can inhibit FOXO3a translocation from cytoplasm to nucleus and downregulate the levels of pro-apoptotic proteins Bim and CC3, leading to the reduction of neuronal apoptosis.

Active Transport, Cell Nucleus ; Animals ; Animals, Newborn ; Apoptosis ; Cell Nucleus ; metabolism ; Female ; Forkhead Box Protein O3 ; metabolism ; Hypoxia-Ischemia, Brain ; pathology ; JNK Mitogen-Activated Protein Kinases ; physiology ; Male ; Neurons ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the role and mechanisms of FOXO3a nuclear translocation in neuronal apoptosis after hypoxia-ischemia (HI).

METHODSOne hundred and sixty 10-day-old Sprague-Dawly rats were randomly divided into two groups: HI and sham-operated. The right common carotid artery was ligated followed by hypoxia exposure for 2.5 hours in the HI group. The sham-operated group rats were not subjected to carotid artery ligation or hypoxia treatment. Rat cerebral cortex was collected at 0.5, 2, 4, 8 and 24 hours after hypoxia. Western blot was used to detect expression of total FOXO3a protein, pnuclear and cytoplasmic FOXO3a and Bim. TUNEL staining was used to detect apoptotic cells.

RESULTSThe nuclear protein of FOXO3a obviously increased from 0.5 to 24 hours after HI in a time-dependent manner compared with the sham-operated group (P<0.01). On the contrary, cytoplasmic protein evidently decreased from 0.5 to 24 hours in the HI group compared with the sham-operated group (P<0.01). Bim protein increased from 0.5 hour, peaked at 2 hours, started to decline at 4 hours (P<0.01), and returned to baseline level at 8 and 24 hours after HI in the HI group compared with the sham-operated group. TUNEL positive cells started to express at 4 hours, and peaked at 24 hours after HI (P<0.01). However, TUNEL positive cells were rarely found in the sham-operated group.

CONCLUSIONSHI induces FOXO3a translocation from cytoplasm to nucleus, and enhances protein expression of its target gene Bim in the neonatal rat brain. The upregulation of Bim expression might be related to neuronal apoptosis.

Animals ; Animals, Newborn ; Apoptosis ; Apoptosis Regulatory Proteins ; analysis ; Bcl-2-Like Protein 11 ; Female ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; physiology ; Hypoxia-Ischemia, Brain ; pathology ; Male ; Membrane Proteins ; analysis ; Neurons ; pathology ; Proto-Oncogene Proteins ; analysis ; Rats ; Rats, Sprague-Dawley

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.
Adenocarcinoma of Lung/pathology* ; Apoptosis/drug effects* ; Cell Cycle Checkpoints/drug effects* ; Cell Cycle Proteins/genetics* ; Cell Line, Tumor ; Forkhead Box Protein O3/physiology* ; Gene Expression Regulation, Neoplastic/drug effects* ; Humans ; Lung Neoplasms/pathology* ; Oligopeptides/pharmacology* ; Proto-Oncogene Proteins c-akt/physiology* ; Up-Regulation