[Corrigendum] Effects of simvastatin on iNOS and caspase­3 levels and oxidative stress following smoke inhalation injury.

After the publication of the above paper, the authors have noticed that the affiliations were presented incorrectly; essentially, Drs Rong­qiang Yang, Peng­fei Guo, Qing­nan Meng, Ya Gao, Imran Khan, Xiao­bo Wang and Zheng­jun Cui are based at the Department of Burn and Repair Reconstruction Surgery, The First Affiliated Hospital of Zhengzhou University, whereas Drs Zhao Ma and Cheng Chang are located at The School of Basic Medical Science of Zhengzhou University. Therefore, the affiliations for this paper should have appeared as follows: Rong­Qiang Yang1, Peng­Fei Guo1, Zhao Ma2, Cheng Chang2, Qing­Nan Meng1, Ya Gao1, Imran Khan1, Xiao­Bo Wang1 and Zheng­Jun Cui1. 1Department of Burn and Repair Reconstruction Surgery, The First Affiliated Hospital of Zhengzhou University; 2The School of Basic Medical Science of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China. The authors regret that these errors with the author affiliations were not noticed prior to the publication of their paper, and apologize for any inconvenience caused. [the original article was published in Molecular Medicine Reports 22: 3405-3417, 2020; DOI: 10.3892/mmr.2020.11413].

Effects of simvastatin on iNOS and caspase­3 levels and oxidative stress following smoke inhalation injury.

The overexpression of inducible nitric oxide synthase (iNOS) induces cell apoptosis through various signal transduction pathways and aggravates lung injury. Caspase­3 is an important protein in the apoptotic pathway and its activation can exacerbate apoptosis. Simvastatin, a hydroxymethyl glutaryl­A reductase inhibitor, protects against smoke inhalation injury by inhibiting the synthesis and release of inflammatory factors and decreasing cell apoptosis. Following the establishment of an animal model of smoke inhalation injury, lung tissue and serum were collected at different time points and the protein and mRNA expression of iNOS and caspase­3 in lung tissue by immunochemistry, western blot and reverse transcription­quantitative polymerase chain reaction, the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in lung tissue and serum were analyzed using thiobarbituric acid method and the WST­1 method. The results were statistically analyzed. The lung tissues of the rats in the saline group and the low­, middle­ and high­dose groups exhibited clear edema and hemorrhage, and had significantly higher pathological scores at the various time points compared with the rats in the control group (P<0.05). Furthermore, lung tissue and serum samples obtained from these four groups had significantly higher mRNA and protein expression levels of iNOS and caspase­3 (P<0.05), significantly lower SOD activity and higher MDA content (P<0.05). Compared with the saline group, the low­, middle­ and high­dose groups had significantly lower pathological scores (P<0.05), significantly lower mRNA and protein expression levels of iNOS, caspase­3 and MDA content in lung tissues (P<0.05) and significantly higher SOD activity in lung tissues and serum. The middle­ and high­dose groups had significantly lower pathological scores (P<0.05), significantly decreased iNOS and caspase­3 mRNA and protein expression in lung tissues, significantly higher SOD activity in lung tissues and serum and a significantly lower MDA content (P<0.05) compared with the low­dose group. With the exception of SOD activity in lung tissues at 24 and 72 h and MDA content in serum at 48 h, no significant differences were observed between the middle­ and high­dose groups. The present study demonstrated that there was an association between the therapeutic effect and dosage of simvastatin within a definitive range. In rats with smoke inhalation injury, simvastatin inhibited iNOS and caspase­3 expression in lung tissues and mitigated oxidative stress, thereby exerting a protective effect. In addition, the effect and dose were associated within a definitive range.

Distinct PD-L1/PD1 Profiles and Clinical Implications in Intrahepatic Cholangiocarcinoma Patients with Different Risk Factors.

Rationale: PD1/PD-L1 immune checkpoint inhibitors have shown promising results for several malignancies. However, PD1/PD-L1 signaling and its therapeutic significance remains largely unknown in intrahepatic cholangiocarcinoma (ICC) cases with complex etiology. Methods: We investigated the expression and clinical significance of CD3 and PD1/PD-L1 in 320 ICC patients with different risk factors. In addition, we retrospectively analyzed 7 advanced ICC patients who were treated with PD1 inhibitor. Results: The cohort comprised 233 patients with HBV infection, 18 patients with hepatolithiasis, and 76 patients with undetermined risk factors. PD-L1 was mainly expressed in tumor cells, while CD3 and PD1 were expressed in infiltrating lymphocytes of tumor tissues. PD1/PD-L1 signals were activated in tumor tissues, and expression was positively correlated with HBV infection and lymph node invasion. More PD1+ T cells and higher PD-L1 expression were observed in tumor tissues of ICC patients with HBV infection compared to patients with hepatolithiasis or undetermined risk factors. More PD1+ T cells and/or high PD-L1 expression negatively impacted the prognosis of patients with HBV infection but not those with hepatolithiasis. Multivariate analysis showed PD1/PD-L1 expression was an independent indicator of ICC patient prognosis. Advanced ICC patients with HBV infection and less PD1+ T cells tended to have good response to anti-PD1 therapy. Conclusion: Hyperactivated PD1/PD-L1 signals in tumor tissues are a negative prognostic marker for ICCs after resection. HBV infection- and hepatolithiasis-related ICCs have distinct PD1/PD-L1 profiles. Further, PD1+ T cells could be used as a biomarker to predict prognosis and assay the efficiency of anti-PD1 immunotherapy in ICC patients with HBV infection.

[Experimental study on the effect of vascular endothelial growth factor 165 gene on vascularization of dermal substitute].

OBJECTIVE: To investigate the effect of vascular endothelial growth factor 165 (VEGF 165) gene on vascularization of dermal substitute in vivo. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in M199 medium containing FBS in the volume fraction of 10% (briefly called complete medium). (1) HUVECs were divided into non-transfection group (without transfection), empty vector group [transfected with pIRES2-enhanced green fluorescent protein (EGFP) plasmid], and VEGF plasmid group (transfected with pIRES2-EGFP-VEGF plasmid) according to the random number table, with 6 wells in each group. At post transfection hour (PTH) 24, the expression of green fluorescent protein (GFP) in each group was observed under inverted phase contrast fluorescence microscope, and the expression rate of GFP was detected with flow cytometer. Cells in non-transfection group were tested with the same methods as listed above. The cells in stable transfection in empty vector group and VEGF plasmid group were sifted by neomycin. The mRNA and protein expression levels of VEGF 165 in cells and the protein amount of VEGF 165 in the supernatant of cell culture medium in 3 groups were respectively determined by real-time fluorescent quantitation PCR, Western blotting, and enzyme-linked immunosorbent assay. (2) Forty-eight male nude mice were divided into 4 groups according to the random number table, with 12 mice in each group. Mice in saline group were subcutaneously implanted with dermal substitutes which had been cultured in saline for 2 days on both sides of back (the same site below); mice in medium group were subcutaneously implanted with dermal substitutes which had been cultured in complete medium for 2 days; mice in non-transfected cells group were subcutaneously implanted with dermal substitutes that had been cultured in complete medium with non-transfected HUVECs for 2 days; mice in transfected cells group were subcutaneously implanted with dermal substitutes that had been cultured in complete medium with HUVECs stably transfected with VEGF plasmid for 2 days. The dermal substitutes in every group were taken out on post operation day (POD) 3, 7, 14, and 21. Distributions of microvessels and HUVECs in dermal substitutes were observed by immunohistochemical staining, and the microvessel number was counted on POD 14; the expression level of VEGF 165 protein in dermal substitutes was determined by Western blotting. The experiments were all done in triplicate. Data were processed with one-way analysis of variance and LSD method. RESULTS: (1) Obvious green fluorescence was only observed in the two groups with transfected cells at PTH 24. Expression rates of GFP in the cells of non-transfection group, empty vector group, and VEGF plasmid group were respectively 0, (85.2 ± 3.2) %, and (93.1 ± 2.4) %. In the non-transfection group, empty vector group, and VEGF plasmid group, the relative expression amounts of VEGF 165 mRNA were respectively 1, 1.05 ± 0.09, and 3.02 ± 0.13 (F = 5.28, P < 0.05); the relative expression amounts of VEGF 165 protein were respectively 0.78 ± 0.16, 0.76 ± 0.13, and 1.92 ± 0.18 (F = 7.62, P < 0.05); the protein quantity of VEGF 165 in cell supernatant was respectively (62.4 ± 2.7), (73.1 ± 3.8), (117.5 ± 3.1) pg/mL (F = 15.08, P < 0.05). The mRNA and protein levels of VEGF 165 and VEGF 165 protein amount in supernatant were significantly higher in VEGF plasmid group than in the other two groups, with P values all below 0.05. (2) The number of HUVECs in dermal substitutes of transfected cells group was significantly higher than that of the other three groups on POD 14. The numbers of microvessels of dermal substitutes on POD 14 in saline group, medium group, non-transfected cells group, transfected cells group were respectively 4.2 ± 1.1, 5.2 ± 1.1, 6.6 ± 0.9, 13.8 ± 0.8 per 200 times visual field (F = 17.96, P < 0.01). The microvessel number in transfected cells group was significantly higher than that of the other three groups, with P values all below 0.05. The relative expression ratio of VEGF 165 protein of dermal substitutes in transfected cells group was significantly higher than that in saline group as of POD 7. On POD 14 and 21, the relative expression ratios of VEGF 165 proteins in non-transfected cells group (1.652 ± 0.086, 2.152 ± 0.062) and transfected cells group (2.403 ± 0.091, 2.879 ± 0.047) were significantly higher than those of saline group (1.299 ± 0.027, 1.362 ± 0.103), with P values all below 0.05. And the index level of transfected cells group was significantly higher than that in non-transfected cells group (with P values below 0.05). The VEGF 165 protein content in dermal substitutes increased with time extension in all groups. CONCLUSIONS: Transfection of VEGF 165 gene in HUVEC could effectively facilitate vascularization of dermal substitutes in vivo by high expression of VEGF 165 protein.