The combination of tetramethylpyrazine and vitamin A acid in the treatment of skin photoaging by activating the HIF-1α pathway.

Skin photoaging is a skin degenerative disease that causes patients to develop malignant tumors. The existing clinical treatment of photoaging has limitations. This greatly reduces the recovery rate of photoaging patients. Studies have confirmed that Ligusticum wallichii Franch (LWF) monomer tetramethylpyrazine (TMP) alleviates various skin diseases. The combination of traditional Chinese medicine and Western medicine helps with this process. Our research aimed to explore the specific treatment mode and molecular mechanism of TMP in treating skin photoaging. CCK-8 assays were used to evaluate the activity and toxicity of HaCaT cells. ß-galactosidase aging, Carbonyl compound and nitrosylated tyrosine assays were used to analyze the aging of HaCaT cells. ROS assays and ELISA were used to analyze the enrichment of ROS. The molecular docking experiment analyzed the binding of TMP and HIF-1α. qRT-PCR and Western blot were used to detect the activation of skin aging-related pathways. HE staining was used to analyze the thickness of the stratum corneum skin on the back skin of mice. 200µg/L LWF alleviates cellular photoaging and mouse skin photoaging by reducing ROS enrichment. Its monomer TMP plays an important role in this process. The combination of TMP and HIF-1α accelerates the degradation of ROS by activating the Nrf2/ARE signaling pathway. This process reduces the apoptosis of cells damaged by light. In addition, we also found that the combination of TMP and retinoic acid (RA) is more beneficial for the treatment of skin damage caused by light in mice. The combination therapy of TMP and RA alleviates skin oxidative stress response through overexpression of HIF-1α. This plan is beneficial for the treatment of skin photoaging.

The expression of ß-Defensin-2, IL-22, IL-22R1 and IL-10R2 in rat model of Klebsiella pneumonia and their correlation with histological grades.

Backgrounds and Aims:Klebsiella pneumoniae represents the most common opportunistic pathogen contributing to Klebsiella pneumonia in hospital-acquired infections. Klebsiella pneumonia has a rapidly progressive clinical course and multi-drug resistant (MDR). Identification of the effective biochemical markers is crucial for improving early diagnosis and treatment of Klebsiella pneumonia. The aims of our study are to 1) investigate the expression of ß-Defensin-2(rßD2), IL-22, IL-22R1 and IL-10R2 in Klebsiella pneumonia-infected rats and 2) their association with the histological grades of Klebsiella pneumonia.Methods and Materials: Fifty specific pathogen free (SPF) male SD rats were randomly divided into two groups: control group (treated with normal saline) and pneumonia group (treated with K. pneumoniae). All animals were sacrificed 1 h, 12 h, 1 d, 3 d, 5 d post infection. The severity and property of pneumonia was evaluated by histopathologic observation and pathogen identification. The mRNA expression of rßD2, IL-22, IL-22R1 and IL-10R2 was measured by RT-qPCR assay. The expression of rßD2 in rat lung tissue was determined by Western blot analysis, and the level of IL-22 in rat serum was determined by ELISA.Results: Histopathologic examination and bacterial counting of lung tissues confirmed the successful establishment of rat pneumonia model. The gene expression of rßD2, IL-22, IL-22R1 and IL-10R2 in pneumonia rats were significantly higher than those in healthy control mice (P < 0.05). The expression of rßD2 was correlated with histological grades of Klebsiella pneumonia and the level of IL-22. RT-qPCR results showed that the peak expression of IL-22R1 appeared earlier than IL-10R2 in rat pneumonia model.Conclusions: The expression of rßD2 and IL-22 was increased significantly at early stage in rat Klebsiella pneumonia model, suggesting that IL-22 and rßD2 might serve as potential biomarkers for the early diagnosis of Klebsiella pneumonia.

HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A.

MicroRNAs (miRNAs) encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2 (HSV-2) infection. In this study, miRNA-H4-5p and miRNA-H4-3p were ectopically expressed in HeLa cells to explore potential cellular targets of viral miRNAs and demonstrate their potential biological functions. The results showed that miRNA-H4-5p could reverse apoptosis induced by actinomycin D (Act-D) and promote cell cycle progression, but miRNA-H4-3p had no such obvious functions. Bioinformatics analysis, luciferase report assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blotting demonstrated that miRNA-H4-5p could bind to the 3'-untranslated region (UTR) of cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase-like 2 (CDKL2) to negatively regulate their expression. We verified that these two targeted genes were associated with cell apoptosis and cell cycle. Furthermore, in HeLa cells infected with HSV-2, we detected significantly reduced expression of CDKN2A and CDKL2 and demonstrated the negative regulation effect of miRNA-H4-5p on these two target genes. Our findings show that viral miRNAs play a vital role in regulating the expression of the host's cellular genes that participate in cell apoptosis and progression to reshape the cellular environment in response to HSV-2 infection, providing further information on the roles of encoded herpesvirus miRNAs in pathogen-host interaction.

Activating transcription factor 4 mediates a multidrug resistance phenotype of esophageal squamous cell carcinoma cells through transactivation of STAT3 expression.

Multidrug resistance (MDR) is a major challenge to the clinical treatment of esophageal cancer. The stress response gene activating transcription factor 4 (ATF4) is involved in homeostasis and cellular protection. However, relatively little is known about the expression and function of ATF4 in esophageal squamous cell carcinoma (ESCC) MDR. In this study, we investigate the potential role and mechanisms of ATF4 in ESCC MDR. We demonstrated that overexpression of ATF4 promotes the MDR phenotype in ESCC cells, while depletion of ATF4 in the MDR ESCC cell line induces drug re-sensitization. We also demonstrated that ATF4 transactivates STAT3 expression by directly binding to the signal transducers and activators of transcription 3 (STAT3) promoter, resulting in MDR in ESCC cells. Significantly, inhibition of STAT3 by small interfering RNA (siRNA) or a selective inhibitor (JSI-124) reintroduces therapeutic sensitivity. In addition, increased Bcl-2, survivin, and MRP1 expression levels were observed in ATF4-overexpressing cells. In conclusion, ATF4 may promote MDR in ESCC cells through the up-regulation of STAT3 expression, and thus is an attractive therapeutic target to combat therapeutic resistance in ESCC.

Enforced expression of microRNA-21 influences the replication of varicella-zoster virus by triggering signal transducer and activator of transcription 3.

Varicella-zoster virus (VZV) causes chronic pain and serious complications, including zoster paresis. However, the mechanism of VZV replication, a critical part of VZV pathogenesis, remains largely unknown and was investigated in the present study. The upregulation of microRNA-21 (miR-21) was identified following VZV infection in vitro by quantitative polymerase chain reaction. The hypothesis that the overexpression of miR-21 activates the signal transducer and activator of transcription 3 (STAT3) signaling pathway was validated by measuring the mRNA expression levels of STAT3 and the anti-apoptotic protein survivin in human malignant melanoma (MeWo) and human embryonic lung fibroblast (HELF) cell lines transfected with miR-21-mimic and comparing them with those in cells transfected with miR-control. To further study the interaction of miR-21, STAT3 and VZV replication, the effects of miR-21 overexpression and STAT3 knockdown were evaluated. Higher virus titers were detected when miR-21 was upregulated in vitro. Moreover, it was identified that significantly lower virus titers were present in MeWo cells in which STAT3 was knocked down. In addition, the overexpression of miR-21 did not stimulate VZV replication in the MeWo cell line when the STAT3 gene was silenced. Therefore, the observations of the present study indicate that the enforced expression of miR-21 promotes the replication of VZV by activating STAT3 in vitro.

[Expression of herpes simplex virus type 2 latency associated transcript ORF1 and its anti-apoptotic function].

To study the expression of herpes simplex virus type 2 latency-associated transcript (LAT) open reading frame 1 (ORF1) and its anti-apoptosis function induced by actinomycin D in Vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, and the expression of ORF1 was identified by RT-PCR. The changes of Vero cells morphology induced by actinomycin D were observed by fluorescence microscopy, Hochest33258 fluorescence staining. Cells viability was evaluated by MTT assay and cells apoptosis rate was detected by flow cytometry. Double digestion and sequencing confirmed the pEGFP-ORF1 was constructed successfully, RT-PCR showed that the target gene was highly expressed in Vero cells. Hochest33258 staining reaveals that Vero cells transfected with pEGFP-ORF1 and induced apoptosis by actinomycin D had no changes in morphology. MTT assay showed that the viabilities of Vero cells transfected with recombinant plasmid pEGFP-ORF1 and induced apoptosis by actinomycin D has no statistically significant difference compared with the untreated normal control group (P > 0.05), but remarkable higher than Vero cells transfected with empty plasmid pEGFP-C2 and induced apoptosis by actinomycin D, the difference was statistically significant (P < 0.05). Flow cytometry assay shows that the cells apoptosis rate had no significant difference between pEGFP-ORF1 group and the normal group, but the cells apoptosis rate ofpEGFP-ORF1 was lower than the pEGFP-C2 group. HSV-2 LAT ORF1 gene can be expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.

[Changes of microRNA expression profiles in Vero cells induced by HSV-2 LAT overexpression].

OBJECTIVE: To investigate the changes in the microRNA expression profile of Vero cells induced by HSV-2 LAT overexpression. METHODS: The full-length open reading frame of HSV-2 LAT was synthesized and cloned into pRetroQ- AcGFP1-C1 vector, and the recombinant retrovirus expressing HSV-2 LAT was packaged. Using a microRNA microarray, the microRNA expression profile changes in Vero cells were analyzed after infection with the recombinant retrovirus. RESULTS: In Vero cells infected with the recombinant retrovirus for stable HSV-2 LAT overexpression, 5 microRNAs (hsa-miR-23a*, kshv-miR-K12-3, hsa-miR-943, hsa-miR-634, and hsa-miR-1270) were up-regulated and 5 (hsa-miR-181a-2*, hsa-miR-450b-5p, hsa-miR-31, hsa-miR-24, and kshv-miR-K12-12*) were down-regulated. CONCLUSION: The expression of HSV-2 LAT can induce changes in microRNA expression profile in Vero cells.

Knowledge-based virtual screening of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus genome.

A novel knowledge-based method is developed to virtually screen potential HLA-A*0201 binders from large-scale peptide candidates. This method utilizes the information from both the crystal structures and experimental affinities of various peptides bound with HLA-A*0201 to construct a single-position mutation free energy profile for accurately characterizing HLA-A*0201-peptide interaction and for effectively predicting the binding affinities of peptides to HLA-A*0201. We employ this method to analyze physicochemical properties and structural implication underlying the specific recognition and association between the HLA-A*0201 and a large panel of peptide segments generated from the herpes simplex virus type 1 (HSV-1) genome, and to evaluate the binding potencies of these peptide candidates to HLA-A*0201. As a result, 288 out of 38,020 candidates are predicted as the potential high-affinity binders of HLA-A*0201, from which three most promising peptides are picked out for further development of potent vaccines against HSV-1. In addition, we also demonstrate that this newly proposed method can successfully identify 8 known binders and 3 known nonbinders from the glycoproteins D and K of HSV-1.

Stepwise identification of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus type 1 genome boosted by a StepRank scheme.

Identification of immunodominant epitopes is the first step in the rational design of peptide vaccines aimed at T-cell immunity. To date, however, it is yet a great challenge for accurately predicting the potent epitope peptides from a pool of large-scale candidates with an efficient manner. In this study, a method that we named StepRank has been developed for the reliable and rapid prediction of binding capabilities/affinities between proteins and genome-wide peptides. In this procedure, instead of single strategy used in most traditional epitope identification algorithms, four steps with different purposes and thus different computational demands are employed in turn to screen the large-scale peptide candidates that are normally generated from, for example, pathogenic genome. The steps 1 and 2 aim at qualitative exclusion of typical nonbinders by using empirical rule and linear statistical approach, while the steps 3 and 4 focus on quantitative examination and prediction of the interaction energy profile and binding affinity of peptide to target protein via quantitative structure-activity relationship (QSAR) and structure-based free energy analysis. We exemplify this method through its application to binding predictions of the peptide segments derived from the 76 known open-reading frames (ORFs) of herpes simplex virus type 1 (HSV-1) genome with or without affinity to human major histocompatibility complex class I (MHC I) molecule HLA-A*0201, and find that the predictive results are well compatible with the classical anchor residue theory and perfectly match for the extended motif pattern of MHC I-binding peptides. The putative epitopes are further confirmed by comparisons with 11 experimentally measured HLA-A*0201-restrcited peptides from the HSV-1 glycoproteins D and K. We expect that this well-designed scheme can be applied in the computational screening of other viral genomes as well.

Secretory expression of human ScFv against keratin in Pichia pastoris and its effects on cultured keratinocytes.

We have got the humanized antibody with high affinity and specificity against keratin by phage antibody library technology. To improve protein yields and get high affinity and specific anti-keratin antibody, we chose to express it in Pichia pastoris. Anti-keratin ScFv gene from plasmid p3MH/ScFv was subcloned into vector pPIC9K. After confirmed by DNA sequence analysis, the recombinant plasmid pPIC9K/ScFv was transduced into the genome of GS115 P. pastoris. Mut(s) multiple insert transformants were screened by G418 and induced by 5 mL/L methanol to express soluble ScFv. After 6 days of methanol induction, anti-keratin ScFv was efficiently secreted into the medium. Western blot and ELISA assay proved the expressed protein had specific keratin-binding activity. After purification, we examined its effect on cultured keratinocytes by Cell cycle analysis, Which indicated that human ScFv against keratin 17 can inhibit the proliferation of keratinocytes by influence the synthesize of keratinocyte DNA. The successful expression of anti-keratin ScFv in P. pastoris laid a solid foundation for its further application.

[Construction and expression of HSV-2gD-Hsp70 fusion protein gene].

To construct and express Hsp70-HSV2gD fusion protein. Genes of Hsp70 and HSV-2gD were subcloned into vectors pGEX-4T-1 respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pGEX-4T-HSP-gD was transformed into E. coli DH5alpha and induced to express with IPTG. The expressed protein was characterized by SDS-PAGE and Western blot after purified. BALB/c mice were immunized with fusion proteins respectively via intra-m uscular injection. The proliferation of spleen lymphocytes, the level of y-IFN in culture and anti-HSV-2gD IgG antibody in serum was detected was detected. The expressed protein was analyzed by SDS-PAGE after induced with IPTG, which showed a new band with an apparent molecular mass corresponding to the predicted size (118 kD). Western Blotting analysis demonstrates that the purified Hsp70-HSV2gD fusion protein had specific binding activity. The stimulation indexes of spleen lymphocytes, the level of gamma-IFN in culture and anti-HSV-2gD IgG antibody in serum of GST-Hsp70-gD group was obviously higher than that of other groups (P < 0.05 respectively). The successful expression of the Hsp70-HSV2gD fusion protein, which can induce immune responses, laid a solid foundation for its further research.

[Expression of anti-keratin human Fab in Pichia pastoris and optimization of expression condition].

AIM: To express human anti-keratin Fab in Pichia pastoris secretively and optimize the expression condition. METHODS: Genes of kappa chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/Fab were subcloned into vectors pPIC9K and pPICZalphaA respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/L and pPICZalphaA/Fd were transducted into the genome of GS115 Pichia pastoris using two-step integrating technology. Mut(+) multiple insert transformants were screened by G418 and Zeocin. The pH value and methanol concentration was adjusted to optimize the expression condition. RESULTS: Under the optimized expression condition, the Fab of anti-keratin antibody was efficiently secreted into the medium. Western blot assay proved that the expressed protein had specific keratin binding activity. CONCLUSION: The successful expression of the anti-keratin Fab in Pichia pastoris has laid a solid foundation for its further application.

[Construction and expression of eukaryotic expression vector for intact IgG against human keratin].

AIM: To construct an eukaryotic expression vector containing the gene of anti-keratin antibody and express it in CHO(dhfr(-)) cells. METHODS: Both the V(L) and V(H) genes of an anti-keratin Fab from a phage display library were amplified by PCR. Using PCR product as the template, the V(kappa) and V(H) genes with the leader sequences (named L(kappa) and L(H) respectively) were amplified by overlapping PCR. After digested with endonuclease Xba I/BamH I and Xho I/Hind III, L(kappa) and L(H) genes were inserted into pWD digested with Xba I/BamH I and Xho I/Hind III, respectively to construct pWDkappaH. After PCR and sequencing, the expression plasmid pWDkappaH was transfected into CHO (dhfr(-)) cells. The culture supernatant of the transfected cells was collected and assayed for IgG activity. RESULTS: The eukaryotic expression vector pWDkappaH was constructed successfully and expressed in CHO(dhfr(-)) cells. The expression of intact IgG against keratin was identified by ELISA and RT-PCR. CONCLUSION: The successful expression of intact IgG against keratin lays the foundation for its clinical application.

[Expression of human Fab antibody against keratin in E.coli and its renaturation].

AIM: To express Fd fragment and L chain of human anti-keratin Fab in E.coli BL21(DE3) respectively and obtain human anti-keratin Fab by renaturation in-vitro. METHODS: Genes of L chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/ Fab were subcloned into vector pET32a respectively. The recombinant plasmids pETL and pETFd were transformed into E.coli BL21(DE3) and induced to express with IPTG. Fd fragment and L chain inclusion bodies were solubilized and combined in equal molar ratio in the refolding solution. The renatured Fab was characterized by SDS-PAGE, Western blot and ELISA. RESULTS: Fd fragment and L chain of human anti-keratin Fab were efficiently expressed. ELISA and Western blot showed that the renatured Fab could bind with human keratin. CONCLUSION: The successfully prepared anti-keratin Fab with binding activity to human keratin laid a solid foundation for its further application.