ObjectiveTo establish an ischemia-reperfusion model in cynomolgus macaques and to analyse the effects of edaravone intervention. MethodsA total of fifteen adult male cynomolgus macaques were randomly divided into three groups: sham operation (Sham group, n=3), ischemia-reperfusion model (Model group, n=6) and edaravone treatment (Edaravone group, n=6). Ischemic-reperfusion model of cynomolgus macaques was established by clamping the M1 branch of the left cerebral artery for 1 h. After 2 h of reperfusion, the animals in Edaravone group were injected with 0.5 mg/kg edaravone intravenously for intervention treatment, while the animals in Sham and Model groups were injected with an equal volume of normal saline intravenously, twice a day, from the 2nd to 7th day. The behavioral video recordings, clinical observations and neurological deficit scores of cynomolgus macaques were obtained, and brain edema volume and cerebral ischemia volume were statistically analyzed. ResultsCompared with the Sham group, the animals in Model group showed typical symptoms of ischemic stroke, with a significant increase in the neurological deficit score， the volumes of edema and infarct of brain tissue (all P＜0.01). Compared with Model group, the neurological deficit score, the volumes of edema and infarct of brain tissue were significantly reduced in Edaravone group (all P＜0.05). ConclusionAn animal model of ischemia-reperfusion in cynomolgus macaques was successfully established, and edaravone was confirmed to alleviate the damage caused by ischemia-reperfusion.

A 73-year-old male patient with right lung squamous cell carcinoma developed edema of bilateral lower limbs, poor appetite, and oliguria after targeted treatment with afatinib 30 mg once daily orally for 80 days. The laboratory tests showed serum creatinine 658 mmol/L, blood urea 26.8 mmol/L, urine protein (++++), and urine occult blood (+++). Renal biopsy showed renal tubular injury, some cells appearing as crescents, and mild IgA deposition. Acute kidney failure was diagnosed, which was considered to be caused by afatinib. Afatinib was stopped and symptomatic treatments including hemodialysis, glucocorticoids, anticoagulants, diuretics, etc. were given. Twelve days later, the edema in both lower limbs was alleviated;19 days later, his daily urine volume was approximately 800 ml; 99 days later, his renal function indicators tended to be normal. The patient did not receive targeted treatment again.

A 73-year-old male patient with right lung squamous cell carcinoma developed edema of bilateral lower limbs, poor appetite, and oliguria after targeted treatment with afatinib 30 mg once daily orally for 80 days. The laboratory tests showed serum creatinine 658 mmol/L, blood urea 26.8 mmol/L, urine protein (++++), and urine occult blood (+++). Renal biopsy showed renal tubular injury, some cells appearing as crescents, and mild IgA deposition. Acute kidney failure was diagnosed, which was considered to be caused by afatinib. Afatinib was stopped and symptomatic treatments including hemodialysis, glucocorticoids, anticoagulants, diuretics, etc. were given. Twelve days later, the edema in both lower limbs was alleviated;19 days later, his daily urine volume was approximately 800 ml; 99 days later, his renal function indicators tended to be normal. The patient did not receive targeted treatment again.

A 65-Year-old male patient with squamous cell lung cancer received chemotherapy with paclitaxel (210 mg by an IV infusion on day 1) and carboplatin (0.5 g by an IV infusion on day 1; 21 days was a cycle) and targeted therapy with apatinib (0.25 g orally once daily). About 1 hour after the first oral administration of apatinib, the patient developed dyspnea, chest tightness, followed by limb numbness and fatigue. About 1 hour after the second administration, the above symptoms recurred, accompanied by lower limb pain, speak disability, and arrhythmia. The symptoms were considered to be related to apatinib. The drug was stopped and the treatments including noninvasive ventilator assisted breathing, antiallergic, antispasmodic, and antiasthmatic were given. Five days later, the above symptoms disappeared.

A 65-Year-old male patient with squamous cell lung cancer received chemotherapy with paclitaxel (210 mg by an IV infusion on day 1) and carboplatin (0.5 g by an IV infusion on day 1; 21 days was a cycle) and targeted therapy with apatinib (0.25 g orally once daily). About 1 hour after the first oral administration of apatinib, the patient developed dyspnea, chest tightness, followed by limb numbness and fatigue. About 1 hour after the second administration, the above symptoms recurred, accompanied by lower limb pain, speak disability, and arrhythmia. The symptoms were considered to be related to apatinib. The drug was stopped and the treatments including noninvasive ventilator assisted breathing, antiallergic, antispasmodic, and antiasthmatic were given. Five days later, the above symptoms disappeared.

A 56-year-old male patient with advanced hepatocellular carcinoma received targeted therapy with lenvatinib 12 mg once daily. After 30 days of medication, the patient developed epistaxis without predisposing factors in the morning, which occurred once every half a month and could be stopped after compression. The drug was not stopped. After 97 days of medication, the patient developed abdominal pain. Physical examination showed abdominal tenderness, especially in the upper and middle abdomen, rebound pain, and muscle tension. Abdominal CT examination showed pneumoperitoneum, hydrops, and perforation of hollow organs of abdomen. Lenvatinib was withdrawn and the conservative treatments with fasting, gastrointestinal decompression, anti-infection, acid suppression, and rehydration were given. After 22 days of treatments, his abdominal pain was relieved and the fluid diet was restored. After 26 days of treatments, the patient had exhaust and defecation. After that, the patient took lenvatinib with the same dose as before by himself again for one week, and epistaxis recurred. There was no epistaxis after discontinuation of lenvatinib again. It was considered that epistaxis and perforation of hollow organs might be related to lenvatinib.

A 75-year-old male patient received an IV infusion of lappaconitine hydrobromide for injection 4 mg dissolved into 5% glucose injection 250 ml once daily due to bone metastases of lung cancer. After a single time of lappaconitine treatment, the patient developed muscle tremor mainly in the limbs, which was obvious in the sitting and standing positions and disappeared after lying flat. After 2 times of lappaconitine treatment, the muscle tremor was aggravated and he was unable to take care of himself due to tremor, showing that his hands could not hold things and walking was difficult. No other abnormal signs were found except the increase of muscle tension. The extrapyramidal symptoms (Parkinson-like symptoms) induced by lappaconitine was considered. Then lappaconitine was stopped and other treatments continued. About 24 hours of lappaconitine withdrawal, the patient′s muscle tremor disappeared. After 8 days of observation, his symptoms did not recur.

A 56-year-old male patient with advanced hepatocellular carcinoma received targeted therapy with lenvatinib 12 mg once daily. After 30 days of medication, the patient developed epistaxis without predisposing factors in the morning, which occurred once every half a month and could be stopped after compression. The drug was not stopped. After 97 days of medication, the patient developed abdominal pain. Physical examination showed abdominal tenderness, especially in the upper and middle abdomen, rebound pain, and muscle tension. Abdominal CT examination showed pneumoperitoneum, hydrops, and perforation of hollow organs of abdomen. Lenvatinib was withdrawn and the conservative treatments with fasting, gastrointestinal decompression, anti-infection, acid suppression, and rehydration were given. After 22 days of treatments, his abdominal pain was relieved and the fluid diet was restored. After 26 days of treatments, the patient had exhaust and defecation. After that, the patient took lenvatinib with the same dose as before by himself again for one week, and epistaxis recurred. There was no epistaxis after discontinuation of lenvatinib again. It was considered that epistaxis and perforation of hollow organs might be related to lenvatinib.

A 75-year-old male patient received an IV infusion of lappaconitine hydrobromide for injection 4 mg dissolved into 5% glucose injection 250 ml once daily due to bone metastases of lung cancer. After a single time of lappaconitine treatment, the patient developed muscle tremor mainly in the limbs, which was obvious in the sitting and standing positions and disappeared after lying flat. After 2 times of lappaconitine treatment, the muscle tremor was aggravated and he was unable to take care of himself due to tremor, showing that his hands could not hold things and walking was difficult. No other abnormal signs were found except the increase of muscle tension. The extrapyramidal symptoms (Parkinson-like symptoms) induced by lappaconitine was considered. Then lappaconitine was stopped and other treatments continued. About 24 hours of lappaconitine withdrawal, the patient′s muscle tremor disappeared. After 8 days of observation, his symptoms did not recur.

OBJECTIVETo establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method.

METHODHuman ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed.

RESULTThe recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity.

CONCLUSIONA phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.

Cell Line, Tumor ; Cicer ; chemistry ; metabolism ; Drug Evaluation, Preclinical ; methods ; Estrogen Receptor alpha ; genetics ; metabolism ; Genes, Reporter ; drug effects ; Genetic Vectors ; metabolism ; Genistein ; chemistry ; pharmacology ; Humans ; Luciferases ; drug effects ; metabolism ; Phytoestrogens ; analysis ; pharmacology ; Plant Extracts ; chemistry ; metabolism ; pharmacology ; Plasmids ; drug effects ; metabolism ; Transfection ; methods