AIM:To investigate the molecular mechanism of Xijiao Dihuang decoction combined with Yinqiao powder (XDY) in treating viral pneumonia, and the effects of XDY on TNF-α-induced permeability in pulmonary microvascular endothelial cells (PMVEC) and the role of PKC-SSeCKS pathway involved.METHODS:The electric conductivity method was used to detect transendothelial electrical resistance (TER) of primarily cultured PMVEC on Transwell chamber at different time points to determine the permeability of PMVEC.After pretreatment for 24 h, the activity of PKC, TER, and the expression of SSeCKS at mRNA and protein levels were detected.Laser scanning confocal microscopy was used to observe the location of SSeCKS and construction of F-actin in PMVEC.RESULTS:The permeability of PMVECs induced by TNF-α reached the peak at 24 h.Compared with control group, the TER in TNF-α group was decreased, and the activity of PKC was increased.Compared with TNF-α group, the activity of PKC in TNF-α with PKC inhibitor group and TNF-α with XDY group was decreased, while the TER was increased, without difference from control group.Compared with control group, the mRNA expression of SSeCKS and phospho-SSeCKS was increased in PMVEC of TNF-α group, but decreased in TNF-α with XDY group compared with TNF-α group.In control group, F-actin was mainly located around the nucleus and at cytoplasmic borders of PMVEC, forming the dense peripheral bundle, and SSeCKS was evenly scattered in the cell.In TNF-α group, the dense peripheral bundle of F-actin surrounding the cells almost disappeared, and SSeCKS was concentrated around the nucleus.Compared with TNF-α group, the distribution and the structure of F-actin and SSeCKS nearly returned to normal in TNF-α with XDY group.CONCLUSION:XDY inhibits the activation of PKC signaling pathway in PMVEC caused by TNF-α to reduce the mRNA expression of SSeCKS and the phosphorylation of SSeCKS, thus preventing the deformation of endothelial cells and reducing the permeability of PMVEC.

Objective To study the effect of soluble, refolded, recombinant extracellular domain of the human Fc gamma receptor Ⅱ a (huFcγRⅡa) on the binding of human IgG to cells. Methods Extra-cellular domain of the huFcγRⅡ a gene was amplified from recombinant plasmid pe3huR Ⅱ by PCR and then cloned into pET-28a vector. The recombinant plasmid pETshuR Ⅱ was transformed into E. coli BL21 (DE3) after identified by PCR and doubly digested. The inclusion bodies of fusion protein were extracted and purified by washing, dissolved in 6 mol/L guanidine buffer, and refolded by rapid dilution technique. The refolding protein activity was tested by ELISA and flow cytometry. Results Extraceilular domain of the huFcγRⅡa gene was successfully cloned into pET-28a. The results of SDS-PAGE showed that the molecular mass (Mr) of the expressed protein was 24.8 × 103, and the expression rate was 30%. The purity of recom-binant shuR Ⅱ was up to 90% after washing. ELISA showed that the recombinant shuR Ⅱ was able to bind human IgG in a dose dependent manner, shuRⅡ could competitively inhibit the binding of human IgG to huFcγRⅡa expressed on the surface of COS-7 cells by flow cytometry. Conclusion The results demon-strate that it is possible to obtain large quantities of recombinant shuR Ⅱ which has comparable binding prep-erties to those of the whole membrane bound huFcγR Ⅱ a.

Objective: To investigate the molecular mechanism and identify potential drugs for subthreshold depression (SD), and elucidate the detalied mechanism of Danzhi Xiaoyao powder (DZXY) in SD. Methods: Using RNA-sequencing, we identified differentially expressed genes (DEGs) in leukocytes of SD compared to healthy controls, deciphered their functions and pathways, and identified the hub genes of SD. We also assessed changes in leukocyte transcription factor activity in patients with SD using the TELiS platform. The Connectivity Map database was retrieved to screen candidate drugs for SD. Based on network pharmacology, we elucidated the “multi-component, multi-target, and multi-pathway” mechanism of DZXY in the treatment of SD. Results: We identified 1080 DEGs (padj <0.05 and |log2 (fold change)| ≥ 1 & protein coding) in the leukocytes of patients with SD. These DEGs, including hub genes, were primarily involved in immune and inflammatory response-related processes. Transcription factor activity analysis revealed similarities between the leukocyte transcriptome profile in SD and the conserved transcriptional response to adversities in immune cells. Connectivity Map analysis identified 28 potential drugs for SD treatment, particularly SB-202190 and TWS-119. Constructing the “Direct Compounds-Direct Targets-Pathways” network for DZXY and SD revealed the curative mechanisms of DZXY in SD, primarily including inflammatory response, lipid metabolism, immune response, and other processes. Conclusion These results provide new insights into the characteristics and functional changes of leukocytes in SD, partially illustrate the pathogenesis of SD, and suggest potential drugs for SD. The curative mechanisms of DZXY in SD are also partially elucidated.

Objective To compare the effects of truncated therapy and conventional therapy on the lung tissue inflammation of mice with pneumonia induced by influenza virus,so as to explore the mechanism of truncated therapy superior to conventional therapy and its relationship with inflammatory cascade after vi-ral infections. Methods 192 Balb/c mice were randomly divided into healthy group, model group, con-ventional therapy group and truncated therapy group. Except for the healthy group, the mice of the other three groups were infected with 50μL 30 LD50 mouse lung-adapted influenza virus strain (FM1) by inoc-ulating intranasally. After 1 h of inoculation, healthy group and model group were administered intragas-trically ( i. g. ) distilled water; conventional therapy group was administered i. g. twice daily Yinqiao Powder for the first three days, then Xijiao Dihuang Decoction for the next four days ( totae seven days);truncated therapy group was administered i. g. Xijiaodihuang Decoction twice daily for consecutive seven days. Then the mice were sacrificed by taking the eyeballs on the 2nd, 4th, 6th, and 8th day for sampling and detecting. The WBC count in bronchoalveolar lavage fluid ( BALF) was detected, the levels of IL-1βand IL-18 in the supernatants of lung homogenate were measured by ELISA and NOD-like receptor fam-ily mem NOD-, LRR-and pyrin domain containing 3 ( NLRP3 ) mRNA in the lung tissue were detected by quantitative realtime-PCR. Results Compared with the model group, the WBC counts of BALF, IL-1β, IL-18 and NLRP3 mRNA in truncated therapy group and conventional therapy group decreased( P<0 . 01 ) . WBC counts , IL-1β and IL-18 began to show the remarkable differences from that of model group since the 2nd day, while conventional therapy group didn’ t. On 8th day, WBC count in truncated group was lower significantly than that in the conventional group(P<0. 01). On the 4th day of being in-fected, NLRP3 mRNA of mice lung tissue expressed highly in the model group , while decreased signifi-cantly in the truncated group only. Conclusion The truncated therapy which may inhibit the inflamma-tory reaction induced by innate immunity at the early phase of infection, can prevent the inflammatory cascade, and can truncate the progress of the disease. The potential mechanism is linked to inhibiting the formation of NLRP3 inflammasome, interfering the mature and secretion of IL-1β and IL-18 .