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Objective:To explore the role of tropomyosin 3 (TPM3) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte pyroptosis and fibroblast activation.Methods:Rat cardiomyocytes (H9c2 cells) were treated with H/R method to simulate myocardial ischemia/reperfusion (I/R) injury, and cell proliferation activity was evaluated with cell counting kit-8 (CCK8). The expression of TPM3 mRNA and protein was detected by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting. H9c2 cells with stable TPM3-short hairpin RNA (shRNA) expression were constructed and treated with H/R (hypoxia for 3 hours, and reoxygenation for 4 hours). The expression of TPM3 was measured by RT-qPCR. The expressions of TPM3, pyroptosis-related proteins including caspase-1, NOD-like receptor protein 3 (NLRP3) and Gasdermin family proteins-N (GSDMD-N) were measured by Western blotting. The expression of caspase-1 was also observed by immunofluorescence assay. The levels of human interleukins (IL-1β, IL-18) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) to elucidate the effect of sh-TPM3 on pyroptosis of cardiomyocytes. Rat myocardial fibroblasts were incubated with the above cell supernatant, and the expressions of human collagen Ⅰ, collagen Ⅲ, matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase inhibitor 2 (TIMP2) were detected by Western blotting to determine the effect of TPM3-interfered cardiomyocytes on the activation of fibroblasts under H/R conditions.Results:Compared with the control group, H/R treatment for 4 hours significantly decreased the survival rate of H9c2 cells [(25.81±1.90)% vs. (99.40±5.54)%, P < 0.01], promoted the expression of TPM3 mRNA and protein [TPM3/GAPDH (2 -ΔΔCt): 3.87±0.50 vs. 1, TPM3/β-Tubulin: 0.45±0.05 vs. 0.14±0.01, both P < 0.01], and promoted the expressions of caspase-1, NLRP3, GSDMD-N, and the enhanced release of cytokines IL-1β and IL-18 [cleaved caspase-1/caspase-1: 0.89±0.04 vs. 0.42±0.03, NLRP3/β-Tubulin: 0.39±0.03 vs. 0.13±0.02, GSDMD-N/β-Tubulin: 0.69±0.05 vs. 0.21±0.02, IL-1β (μg/L): 13.84±1.89 vs. 4.31±0.33, IL-18 (μg/L): 17.56±1.94 vs. 5.36±0.63, all P < 0.01]. However, compared with the H/R group, sh-TPM3 significantly weakened the promoting effects of H/R on these proteins and cytokines [cleaved caspase-1/caspase-1: 0.57±0.05 vs. 0.89±0.04, NLRP3/β-Tubulin: 0.25±0.04 vs. 0.39±0.03, GSDMD-N/β-Tubulin: 0.27±0.03 vs. 0.69±0.05, IL-1β (μg/L): 8.56±1.22 vs. 13.84±1.89, IL-18 (μg/L): 9.34±1.04 vs. 17.56±1.94, all P < 0.01]. In addition, the expressions of collagen Ⅰ, collagen Ⅲ, TIMP2, and MMP-2 in myocardial fibroblasts were significantly increased by the cultured supernatants from the H/R group (collagen Ⅰ/β-Tubulin: 0.62±0.05 vs. 0.09±0.01, collagen Ⅲ/β-tubulin: 0.44±0.03 vs. 0.08±0.00, TIMP2/β-tubulin: 0.73±0.04 vs. 0.20±0.03, TIMP2/β-Tubulin: 0.74±0.04 vs. 0.17±0.01, all P < 0.01). However, these boosting effects were weakened by sh-TPM3 (collagen Ⅰ/β-Tubulin: 0.18±0.01 vs. 0.62±0.05, collagen Ⅲ/β-Tubulin: 0.21±0.03 vs. 0.44±0.03, TIMP2/β-Tubulin: 0.37±0.03 vs. 0.73±0.04, TIMP2/β-Tubulin: 0.45±0.03 vs. 0.74±0.04, all P < 0.01). Conclusion:Interference with TPM3 can alleviate H/R-induced cardiomyocyte pyroptosis and fibroblast activation, suggesting that TPM3 may be a potential target of myocardial I/R injury.
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Objective To investigate the effect of Xinmailong injection combined with Qiliqiangxin capsule on serum BNP , NF-kappa B and cystatin C in the treatment of chronic heart failure in the elderly .Methods 78 cases of elderly patients with chronic heart failure were selected and randomly divided into two groups according to the random number table, 39 cases in each group.All patients received basic treatment, the control group received standard treatment, and the study group received more with Xinmailong injection combined with Qiliqiangxin capsule,and levels of serum B-type natriuretic peptide(BNP), cystatin C(CysC) and NF-κB were compared, left ventricular function and 6 min walking test were detected and the clinical curative effect were compared after the treatment.Results Compared with before treatment, levels of serum BNP, CysC and NF-κB in 2 groups decreased(P<0.01), left ventricular ejection fraction(LVEF) increased(P<0.05), left ventricular end-systolic volume(LVESV) reduced(P<0.05), 6 min walking distance increased(P<0.01), scores of symptom decreased(P<0.01), compared with the control group, levels of serum BNP, CysC and NF-κB in study group were lower, LVEF were higher ( P<0.05 ) , levels of LVEDV and LVESV were lower ( P<0.05 ) , scores of 6 min walking distance were higher(P<0.01), scores of symptom were lower (P<0.01), the effective rate was higher(P<0.05).Conclusion Xinmailong injection combined with Qiliqiangxin capsule in the treatment of chronic heart failure in the elderly was effective , and it can improve left ventricular function, the possible mechanism related to the decrease of BNP, CysC and NF-κB levels.
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Objective To investigate the protective effect of electroacupuncture pretreatment on myocardial ischemia/reperfusion (I/R) injury and its influence on high mobility group box 1 (HMGB1) expression in rats. Methods Sixty Wistar rats were randomly divided into sham operation group, myocardial I/R model group and electroacupuncture pretreatment group by random number table (each n = 20). Myocardial I/R injury model was reproduced by ligating the left ventricular branch coronary artery at about 0.5 cm below the atrial appendage lower margin for 10 minutes to occlude the blood flow, then the ligature was relaxed for 1 hour reperfusion; in electroacupuncture pretreatment group, 7 days before I/R, the electroacupuncture at Neiguan acupoint was applied once daily for 20 minutes till the 7th day when I/R was established. Under light microscope, the pathological changes of myocardial specimen stained by hematoxylin-eosine (HE) method were observed. The myocardial histopathological integral was detected by semi quantitative integral method, and the changes of histological scores in three groups were investigated. The levels of plasma HMGB1, tumor necrosis factor-α (TNF-α), cardiac troponin T (cTnT) were detected by enzyme-labeled immunosorbent assay (ELISA). The expressions of HMGB1, monocyte chemotactic protein-1 (MCP-1), TNF-αmRNA and protein in myocardium were detected by reverse transcription-polymerase chain reaction (PT-PCR) and Western Blot. Results Under light microscope, the myocardial tissue in myocardial I/R model group showed partial fracture of myocardial fibers, large patches of myocardial cell necrosis, hazy boundary, cellular condensation, rupture and dissolution or even disappearance, interstitial edema with a lot of inflammatory cell infiltration; the above myocardial tissue injury in electroacupuncture pretreatment group was significantly milder than that in myocardial I/R model group. Compared with sham operation group, in myocardial I/R model group the HMGB1, TNF-α, cTnT contents and histological score were significantly increased [HMGB1 (μg/L):9.64±1.16 vs. 2.15±0.31, TNF-α(μg/L):91±22 vs. 19±5, cTnT (μg/L):1.50±0.35 vs. 0.07±0.03, histological score:2.5±0.3 vs. 0.0±0.0, all P<0.01], HMGB1, MCP-1, TNF-α mRNA and protein expressions were increased obviously (HMGB1 mRNA: 1.42±0.16 vs. 0.02±0.00, MCP-1 mRNA:0.46±0.06 vs. 0.01±0.00, TNF-αmRNA:0.75±0.04 vs. 0.03±0.00;HMGB1 protein:1.08±0.01 vs. 0.20±0.01, MCP-1 protein:0.92±0.03 vs. 0.40±0.01, TNF-αprotein:1.10±0.02 vs. 0.35±0.01, P<0.05 or P<0.01);compared with myocardial I/R model group, in electroacupuncture pretreatment group, HMGB1 (6.58±0.73), TNF-α (63±19), cTnT (1.15±0.31) levels were significantly decreased (all P < 0.01), HMGB1, MCP-1, TNF-αmRNA and protein expressions were markedly reduced (mRNA expression was 0.74±0.12, 0.18±0.02, 0.10±0.03, and protein expression was 0.40±0.01, 0.36±0.02, 0.50±0.02, respectively all P<0.05), and histological score (1.2±1.0) was remarkably lowered (P < 0.01). Conclusion Electroacupuncture pretreatment may reduce the myocardial I/R injury in rats, and the mechanism may be related to the amelioration of inflammatory response mediated by HMGB1 at late stage.
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Objective:To evaluate the feasibility and clinical efficacy of the treatment of lumbar spine steno-sis by modified unilateral approach for bilateral decompression under microendoscope.Method:217 cases with degenerative lumbar spine stenosis from September 2003 to December 2008 were treated by modified unilater-al approach for bilateral decompression of central spinal canal and nerve root canal under microendoscope.A-mong them laminectomy of one segment was done in 163 cases,two segments were done in 54 cases.After surgery,the routine radiograph were carried out and the Nakai criterion was used for assessment.Result:The mean operative time was 48±13min (range,25 to 95min),the average blood loss was 37±9ml (range,25 to 180ml),the average skin incision length was 2.2±0.2cm(1.8 to 2.4cm).2 cases had dural matter tearing,which healed after filling with gelatin foam and 2 weeks bed rest,after that no cerebrospinal fluid leakage was noted after surgery.Mislocation was noted in 1 case.No nerve inury and postoperative infection oceurred.Postoperative CT scan demonstrated complete decompression of the central spinal canal and nerve root canal.All patients were followed-up for an average of 14 months (range,3 months to 24 months).The Nakai criterion at final follow-up showed clinical excellent in 134 cases,good in 63 cases,fair in 16 cases and worse in 4 cases, with the excellent and good rate of 90.8%.No lumbar spine instability was noted.Conclusion:Treatment of de-generative lumbar spine stenosis by modified unilateral approach for bilateral decompression under mieroendo-scope has the merit of minimal invasive,less complications and reliable therapeutical effect.