BACKGROUND: Angiotensin Ⅱ (Ang Ⅱ) can induce cardiac hypertrophy and platelet-derived growth factor(PDGF) also stimulates cardiac hypertrophy. Is AngⅡ responsible for the pathogenesis of cardiac hypertrophy by inducing PDGF receptor expression?OBJECTIVE: To investigate the effect of mitogen activated protein kinase (MAPK) on the role of cardiac hypertrophy induced by Ang Ⅱ in cardiac myocytes so as to provide theoretical basis for clinical prevention and cure of cardiac hypertrophy.DESIGN: Controlled experimental study taking cardiac myocytes of cultured neonatal rats as subjects.SETTING: Department of pathophysiology in a university.MATERIALS: The experiment was completed in the Department of Pathophysiology, Medical College of Peking University. A total of 80 Wistar rats of either gender, aged 1 - 3 days, were provided by the Animal Center of Medical College, Peking University. Their hearts were removed for myocyte culture in the Cell Culture Laboratory.INTERVENTIONS: The cultured neonatal rat cardiac myocytes treated with 10-7mol/L Ang Ⅱ were Ang Ⅱ group, and those preincubated with 10-5mol/L PD98059(an antagonist of MAPK) for 30 minutes and then treated with Ang Ⅱ were PD98059 group. Cardiac myocytes of normal neonatal rats were as control group. The expression of PDGF-β was detected by western blot at 24 hours.MAIN OUTCOME MEASURES: Content of PDGF-β receptor in neonatal rat cardiac myocytes.RESULTS: The expression of PDGF-β receptor induced by Ang Ⅱ at neonatal rat cardiac myocytes markedly increased at 24 hours (432.41 ± 54.08) compared with that of control group(197.65 ± 44. 10) ( q = 6.77, P< 0.01 ). PDGF-β receptor expression of PD98059 group(317.2 ± 21.12) decreased compared with that of Ang Ⅱ group(q = 3.91, P < 0.05) .However, the expression did not return to the level of control group, and there was significant difference between PD98059 group and control group( q= 3.85, P <0.05).CONCLUSION: The results indicate that angiotensin Ⅱ promotes cardiac hypertrophy through inducing expression of PDGF receptor, in which mitogen activated protein kinase participates in. Maybe it is another important mechanism for Ang Ⅱ -induced cardiac hypertrophy. The results can provide experimental data for the primary and secondary prevention in heart rehabilitation.

AIM: To investigate the crosstalk between angiotensin Ⅱ (AngⅡ)-mediated and platelet-derived growth factor (PDGF)-mediated signal transduction in vascular smooth muscle proliferation.METHODS: A model of renal hypertension was made by two kidney/one-clip operation. Level of PDGF receptor ? subunit of aorta was measured by Western Blot analysis. The effect of Ang Ⅱ on PDGF receptor ? subunit expression was investigated in culture rat aortic vascular smooth muscle cells (VSMC).RESULTS: Systolic blood pressure obviously increased at 8th week after operation, whereas the level of PDGF receptor ? subunit of aorta significantly increased by 126.6% ( P

AIM: To investigate the crosstalk between angiotensin Ⅱ (AngⅡ)-mediated and platelet-derived growth factor (PDGF)-mediated signal transduction in vascular smooth muscle proliferation.METHODS: A model of renal hypertension was made by two kidney/one-clip operation. Level of PDGF receptor β subunit of aorta was measured by Western Blot analysis. The effect of Ang Ⅱ on PDGF receptor β subunit expression was investigated in culture rat aortic vascular smooth muscle cells (VSMC).RESULTS: Systolic blood pressure obviously increased at 8th week after operation, whereas the level of PDGF receptor β subunit of aorta significantly increased by 126.6% (P＜0.05) in 2K1C rats compared with control group. The expression of PDGF receptor β subunit in cultured VSMC stimulated by AngⅡ was higher than that of control by 192.74%(P＜0.01). The effect of AngⅡ was inhibited remarkably by pretreated with losartan, a kind of specific AngⅡ receptor 1 (AT1) subtype antagonist and U73122, a kind of phospholipase C inhibitor. The effect was partly blocked by PD98059, which inhibit the activity of mitogen-activated, ERK-activating kinase (MEK).CONCLUSION: AngⅡ-induced PDGF receptor β subunit expression is regulated by the AT1 and its downstream signal molecule-PLC and ERK, might participate in the intracellular signal transduction pathway.

AIM: To investigate the role of G?q/11-mediated signal transduction pathway in cardiac hypertrophy induced by angiotensin II (AngII). METHODS: Renal hypertension was performed by placing a sliver clip around the left renal artery(2K1C). Hemodynamic parameters, the ratio of left ventricular weight to body weight, the AngII contents and the activities of phospholipase C (PLC) in myocardium were measured at 1st, 2nd, 4th and 8th week after operation, respectively. The levels of G?q/11 were assayed by Western blot analysis. -leucine incorporation and levels of G?q/11 were measured in cultured neonatal rats ventricular myocyte after AngII stimulation. RESULTS: In 2K1C group, blood pressure and the ratio of left ventricle of heart to body weight were significantly increased compared with sham group between 2nd to 8th week. AngII content increased from 1st to 8th week after operation. Compared with the sham group, levels of G?q/11 in 2K1C group increased by 25.0% and 35.8% at 4th and 8th week( P

Objective: To establish the Wistar rat model of acute diquat poisoning and observe the pathological damage of main target organs. Methods: Thirty-six Wistar rats were randomly divided into six groups (n=6) , including one normal saline control group and five treatment groups which were separately given single-dose of intragastric administration at the doses of 46.2 mg/kg, 77.0 mg/kg, 115.5 mg/kg, 231.0 mg/kg and 346.5 mg/kg. The pathological changes of lung, liver and kidney were observed by hematoxylin and eosin (HE) and Masson staining. The optimal dose was determined according to the general situation and pathological changes. Thirty-six Wistar rats were randomly divided into five treatment groups and one normal saline control group. Treatment groups were given single-dose of intragastric administration according to the optimal dose. The rats were sacrificed at 1st, 3rd, 7th, 11th and 14th day after exposed, respectively. The activity of serum glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminase (AST) were measured by chemical colorimetry. The pathological changes of lung, liver and kidney were observed by HE and Masson staining. Results: According to 14 d survival rate, the toxic symptoms and pathological changes, 115.50 mg/kg was determined the best dose. Given single-dose of intragastric administration at the doses of 115.50 mg/kg, it was found that the serum AST and ALT activity of rats on the first and third day of exposure was significant higher than those in control group. The results of pathological examination exhibited that in 115.50 mg/kg group, the pathological changes of lung, liver and kidney began to appear on the first day of exposure, the pathological changes were the most serious on the third day, and then gradually alleviated. On the 14th day, the alveolar septum was slightly widened, with inflammatory cell infiltration, local alveolar cavity became narrow, atrophy, peripheral alveolar compensation, bronchi and alveolar septum collagen fiber proliferation; The local renal tubular epithelial cells were enlarged and necrotic; the central vein surrounding hepatic cells showed vacuolar degeneration with punctate necrosis. Conclusion The rat model of acute diquat poisoning can be successfully induced by single-dose of intragastric administration. The condition of wistar rats and the pathological damage of the main target organs could be observed during the whole course of 115.50 mg/kg administration.

Objective:To investigate the mechanism of Wenjing Decoction in the treatment of hepatic fibrosis through network pharmacological methods, and conducting animal experiments to verify the core targets.Methods:The TCMSP database platform was used to screen the active components and related targets of Wenjing Decoction, and the Uniprot database was used to obtain the target genes corresponding to the active components of Wenjing Decoction. The network diagram of "Chinese materia medica-compound-target" was constructed in Cytoscape 3.7.2, and the GeneCards database was used to search liver fibrosis related targets. String database was used to construct a protein interaction network (PPI) to screen the core components and key targets of liver fibrosis, and GO analysis and KEGG pathway enrichment were performed. Animal experiments were conducted to verify the results of the analysis. 10 mice were selected as the blank group, and the remaining 45 rats were induced with carbon tetrachloride induced liver fibrosis model. After modeling, 40 successfully modeled rats were divided into model group and Wenjing Decoction high, medium-, and low-dosage groups using a random number table method, with 10 rats in each group. Wenjing Decoction high, medium-, and low-dosage groups were orally administered with 1.5×3.18, 3.18, and 0.5×3.18 g/kg Wenjing Decoction, respectively. The blank group was orally administered with equal volume distilled water once a day for 8 consecutive weeks. HE staining was used to observe the histopathological and morphological changes in the liver of rats. The serum GPT and GOT levels of rats were detected using a fully automated biochemical analyzer, and the expressions of TNF, AKT, and IL-6 proteins in rat liver tissue was detected using Western Blot.Results:A total of 188 active components of Wenjing Decoction were obtained, and the active components with higher degree values were β-sitosterol, quercetin, naringenin, etc. 799 liver fibrosis gene targets were collected, and the core target genes of the PPI network were TNF, AKT, IL6, etc. The key anti-hepatic fibrosis related pathways were obtained by GO function and KEGG analysis, including pathway in cancer, TNF, PI3K-Akt and other signalling pathways. Results of animal experiments showed that there were obvious inflammatory infiltration, collagen fibre and pseudo lobe generation in the liver tissue of rats in the model group, and the levels of inflammation and fibrosis in the liver tissue of rats in the Wenjing Decoction high, medium-, and low-dosage groups were improved to different degrees compared with that of the model group; compared with the model group, the levels of serum GPT and GOT decreased ( P<0.05); the protein expressions of TNF, AKT and IL6 in the Wenjing Decoction high, medium-, and low-dosage groups decreased ( P<0.05). Conclusion:Wenjing Decoction may exert anti-liver fibrosis effects by intervening in TNF, AKT, IL6 targets, regulating cancer pathways, TNF, PI3K Akt and other signaling pathways.

OBJECTIVETo identify the gene mutation in a Chinese family with congenital long QT syndrome (LQTS) and predict the changes of the secondary structure of the protein.

METHODSPolymerase chain reaction and DNA sequencing were used to screen for KCNH2 mutation in the proband. After the mutation was identified, KCNH2 gene of the family members was screened by multiplex PCR with site-specific primers. Network analysis software was used to predict the secondary structure of the KCNH2 protein.

RESULTSA novel heterozygous missense mutation of F463L(GenBank accession no.EU218526) located at the transmembrane domain S2 of KCNH2 was detected. The mutation did not result in the change of the transmembrane domain, but altered the hydrophobicity and secondary structure of the protein.

CONCLUSIONThe novel mutation identified in this study has enriched the GenBank data of ion channel gene mutation in LQTS. The changes of the secondary structure caused by the gene mutation were analyzed by Mfold and TMHMM software, which may help to understand LQTS.

Adult ; Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; DNA Mutational Analysis ; ERG1 Potassium Channel ; Ether-A-Go-Go Potassium Channels ; chemistry ; genetics ; Female ; Humans ; Hydrophobic and Hydrophilic Interactions ; Long QT Syndrome ; congenital ; genetics ; Male ; Molecular Sequence Data ; Mutation, Missense ; Pedigree ; Protein Structure, Secondary ; Protein Structure, Tertiary

Objective:To investigate the effects of diquat (DQ) on the expression of intestinal pyroptosis-related proteins and tight junction proteins in rats, and to analyze the role of pyroptosis in the intestinal injury of rats with acute DQ poisoning.Methods:A total of 36 Wistar male rats were randomly divided into control group, and 3 hours, 12 hours, 36 hours and 3 days exposure groups, with 6 rats in each group. Each exposure group was given 1/2 median lethal dose (LD50) of 115.5 mg/kg DQ by one-time gavage. The control group was given the same amount of normal saline by gavage. The control group was anesthetized at 3 hours after DQ gavage to take jejunal tissues; each exposure group was anesthetized at 3 hours, 12 hours, 36 hours, and 3 days after DQ gavage to take jejunal tissues, respectively. The general conditions of the rats were recorded. The pathological changes of jejunum tissue were observed by hematoxylin-eosin (HE) staining. The expression of intestinal pyroptosis-related proteins [NOD-like receptor protein 3 (NLRP3), cysteine aspartate-specific protease 1 (caspase-1), Gasdemin D (GSDMD)] in the intestinal tissues was observed by immunohistochemical staining. Western blotting was used to detect the expression of intestinal pyroptosis-related proteins and intestinal tight junction proteins (Occludin and Claudin-1).Results:Light microscopy showed that pathological changes occurred in jejunum tissue at the early stage of exposure (3 hours), and the injury was the most serious in the 12 hours exposure group, with a large number of inflammatory cells infiltrating in the tissue, and the damage was significantly reduced after 3 days exposure. Immunohistochemical results showed that NLRP3, caspase-1 and GSDMD were expressed in the jejunal mucosa of the control group and the exposure groups, and the positive cells in the control group were less expressed with light staining. The expression of the above proteins in the exposed group was increased significantly and the staining was deep. Western blotting results showed that compared with the control group, the expression of NLRP3 protein in jejunum tissues of all groups was increased, with the most significant increase in the 36 hours group (NLRP3/β-actin: 1.47±0.06 vs. 0.43±0.14, P < 0.01). Compared with the control group, the expression of GSDMD protein in the 3 hours, 12 hours and 36 hours exposure groups increased, and the expression of GSDMD protein in the 3 hours and 12 hours exposure groups increased significantly (GSDMD/β-actin: 1.04±0.40, 1.25±0.15 vs. 0.65±0.25, both P < 0.05). The expression of caspase-1 protein was increased in 36 hours exposure group compared with the control group (caspase-1/β-actin: 1.44±0.34 vs. 0.98±0.19, P > 0.05). Compared with the control group, the expression of Occludin and Claudin-1 proteins in each exposure group decreased, and the expression of Occludin proteins was significantly decreased in the 3 hours, 12 hours, and 36 hours exposure groups decreased significantly (Occludin/β-actin: 0.74±0.17, 0.91±0.20, 0.79±0.23 vs. 1.41±0.08, all P < 0.05). Although the protein expression of Claudin-1 decreased in each exposure group, the difference was not statistically significant. Conclusion:The intestinal injury caused by acute DQ poisoning may be related to the activation of pyroptosis pathway of small intestinal cells and the reduction of the density of intercellular junctions.

OBJECTIVE: To observe the toxicokinetic parameters, absorption characteristics and pathomorphological damage in different parts of the gastrointestinal tract of rats poisoned with different doses of diquat (DQ). METHODS: Ninety-six healthy male Wistar rats were randomly divided into a control group (six rats) and low (115.5 mg/kg), medium (231.0 mg/kg) and high (346.5 mg/kg) dose DQ poisoning groups (thirty rats in each dose group), and then the poisoning groups were randomly divided into 5 subgroups according to the time after exposure (15 minutes and 1, 3, 12, 36 hours; six rats in each subgroup). All rats in the exposure groups were given a single dose of DQ by gavage. Rats in the control group was given the same amount of saline by gavage. The general condition of the rats was recorded. Blood was collected from the inner canthus of the eye at 3 time points in each subgroup, and rats were sacrificed after the third blood collection to obtain gastrointestinal specimens. DQ concentrations in plasma and tissues were determined by ultra-high performance liquid chromatography and mass spectrometry (UPHLC-MS), and the toxic concentration-time curves were plotted to calculate the toxicokinetic parameters; the morphological structure of the intestine was observed under light microscopy, and the villi height and crypt depth were determined and the ratio (V/C) was calculated. RESULTS: DQ was detected in the plasma of the rats in the low, medium and high dose groups 5 minutes after exposure. The time to maximum plasma concentration (Tmax) was (0.85±0.22), (0.75±0.25) and (0.25±0.00) hours, respectively. The trend of plasma DQ concentration over time was similar in the three dose groups, but the plasma DQ concentration increased again at 36 hours in the high dose group. In terms of DQ concentration in gastrointestinal tissues, the highest concentrations of DQ were found in the stomach and small intestine from 15 minutes to 1 hour and in the colon at 3 hours. By 36 hours after poisoning, the concentrations of DQ in all parts of the stomach and intestine in the low and medium dose groups had decreased to lower levels. Gastrointestinal tissue (except jejunum) DQ concentrations in the high dose group tended to increase from 12 hours. Higher doses of DQ were still detectable [gastric, duodenal, ileal and colonic DQ concentrations of 6 400.0 (1 232.5), 4 889.0 (6 070.5), 10 300.0 (3 565.0) and 1 835.0 (202.5) mg/kg respectively]. Light microscopic observation of morphological and histopathological changes in the intestine shows that acute damage to the stomach, duodenum and jejunum of rats was observed 15 minutes after each dose of DQ, pathological lesions were observed in the ileum and colon 1 hour after exposure, the most severe gastrointestinal injury occurred at 12 hours, significant reduction in villi height, significant increase in crypt depth and lowest V/C ratio in all segments of the small intestine, damage begins to diminish by 36-hour post-intoxication. At the same time, morphological and histopathological damage to the intestine of rats at all time points increased significantly with increasing doses of the toxin. CONCLUSIONS The absorption of DQ in the digestive tract is rapid, and all segments of the gastrointestinal tract may absorb DQ. The toxicokinetics of DQ-tainted rats at different times and doses have different characteristics. In terms of timing, gastrointestinal damage was seen at 15 minutes after DQ, and began to diminish at 36 hours. In terms of dose, Tmax was advanced with the increase of dose and the peak time was shorter. The damage to the digestive system of DQ is closely related to the dose and retention time of the poison exposure.
Animals ; Male ; Rats ; Diquat/toxicity* ; Gastrointestinal Diseases ; Intestines ; Poisons ; Rats, Wistar ; Toxicokinetics

Objective:To explore the clinical features of acute diquat (DQ) poisoning, and further improve the awareness of acute DQ poisoning.Methods:A retrospective analysis was performed on the clinical data of patients with acute DQ poisoning diagnosed in the emergency department of the Second Hospital of Hebei Medical University from January 1, 2019 to December 31, 2021. The clinical data included age, gender, exposure routes, presence of pesticides (drugs) mixture poisoning, dosage of poison, the time from taking poisoning to admitting in the emergency department, clinical manifestations, laboratory data, treatment, hospital days, prognosis and survival days.Results:The number of cases who firstly complained of acute DQ poisoning in the past three years were 19 cases in 2019, 28 cases in 2020, and 51 cases in 2021. A total of 12 patients were excluded due to being diagnosed paraquat (PQ) poisoning by toxicology detection. Finally, 86 cases of acute DQ poisoning were included, including 80 cases of oral DQ poisoning, 1 case of intramuscular injection, 1 case of binocular contact and 4 cases of dermal exposure. In 80 cases of oral DQ poisoning, there were 70 cases of diquat poisoning alone (42 cases survived, 28 cases died) and 10 cases of pesticide mixture poisoning (6 cases survived, 4 cases died). The time from oral poisoning to admitting in the emergency department was 0.5-96.0 hours, with an average of (8.6±5.8) hours. The time of intramuscular injection poisoning to admitting in the emergency department was 3 hours. The time of dermal exposure to admitting in the emergency department was relatively long, with an average of 66.1 hours. The time from oral simple DQ poisoning to death was 12.0-108.0 hours, and the time from oral mixed DQ poisoning to death was 24.0-576.0 hours. A total of 70 patients with oral diquat poisoning alone presented various degrees of multiple organ injuries. All patients presented gastrointestinal symptoms such as nausea and vomiting. Renal injury and central nervous system injury were the most significant and closely related to the prognosis.Conclusions:Acute oral DQ poisoning can cause to multiple organ injuries, and the clinical manifestations are related to the dose of the poison. In severe cases, acute renal failure and refractory circulatory failure occur within 24 hours after poisoning, and severe central nervous system injury with disturbance of consciousness as the primary manifestation occurs within 36 hours, followed by multiple organ failure until death.