Objective To observe the inhibitory effect of Tirofiban on different shear-induced platelet aggregation, and to provide medication suggestions for the treatment of thrombosis in different hemodynamic environment. Methods Polydimethylsiloxane ( PDMS)-glass microchannel chips were fabricated by soft lithography. The whole blood of healthy volunteers anticoagulated with sodium citrate was collected and incubated with different concentrations of Tirofiban in vitro. The blood flowed through the straight microchannel or channel with 80% narrow for 150 seconds at the speed of 11 μL/ min and 52 μL/ min, respectively. The wall shear stress rates in straight channel at 11 μL/ min and 52 μL/ min were 300 s-1 and 1 500 s-1, respectively. The maximum wall shear rates in the channel with 80% occlusion at 11 μL/ min and 52 μL/ min were 1 600 s-1 and 7 500 s-1, respectively. The adhesion and aggregation images of fluorescent labeled platelets on glass surface were photographed with the microscope, and the fluorescent images were analyzed with Image J. The platelet surface coverage ratio was used as a quantitative index of platelet aggregation behavior, and the IC50 of Tirofiban for platelet inhibition was calculated under different shear rates. Flow cytometry was used to detect the platelet activation index (CD62P, PAC-1) in the whole blood at 52 μL/ min in channel with 80% occlusion. Results Tirofiban inhibited platelet aggregation in a dose-dependent manner, and the inhibitory effect was related to the shear rate. Under the shear rates of 11 μL/ min and 52 μL/ min, the aggregation was almost completely inhibited when the concentration in straight channel reached 100 nmol / L. When the concentration in channels with 80% occlusion reached 1 μmol / L, the aggregation was almost completely inhibited. IC50 values at 11 μL/ min and 52 μL/ min in straight channel were 2. 3 nmol / L and 0. 5 nmol / L, respectively. IC50 values at 11 μL/ min and 52 μL/ min in channels with 80% occlusion were 20. 73 nmol / L and 4. 5 nmol / L. Pathologically high shearforce induced an increase in platelet activation, which could be inhibited by Tirofiban. Conclusions Tirofiban can effectively inhibit shear-induced platelet aggregation, and different concentrations of Tirofiban should be given according to the thrombus formed in different shear force environment in clinic practice

To test the therapeutic effect of recombinant fusion polypeptide hEGF-AWRK6 (EK) on burn infection of model mice. EK6 was expressed and purified with Escherichia coli expression system, and the Ⅱ degree burns and Pseudomonas aeruginosa infection model mouse were established. Experiment group was treated with EK (30 mg/L), and the control group was treated with PBS, gentamicin (30 mg/L), burn ointment (10 mg/L). The wound healing rate and colony count were calculated. Wound and surrounding skin were taken for HE staining and collagen western-blot analysis, and the wound pathological changes were observed after 10 days of drug delivery. The results showed that fusion peptide EK was successfully expressed and purified with significant antibacterial activities against Pseudomonas aeruginosa. Compared to the control group, the colony count (CFU) of the wound surface in EK mouse had a remarkable decrease (P<0.01) and healing rate had a significant increase in group EK6 (P<0.01). Pathological analysis result showed that compared to the control group, wound dermal cells in group EK arranged regularly, had more hair growth and a faster epithelization. These results indicated that the fusion peptide EK would be a good candidate for the drug development for the treatment of burning wounds.

Objective : The purpose of this study was to clarify the regulatory effect and mechanism of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) on human periodontal ligament stem cell (hPDLSC) proliferation and osteogenic differentiation under inflammatory environment and to provide a new target for the treatment of periodontitis. Methods: SHP2 was knocked down in hPDLSCs, and the transfection efficiency of SHP2 was detected by RT-qPCR and Western blot. An in vitro inflammatory environment was created using tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The effect of SHP2 knockdown on hPDLSC viability under normal and inflammatory conditions was detected by CCK-8, and the osteogenic capacity of hPDLSCs under normal and inflammatory conditions was detected by ALP staining, ALP activity, ARS staining, RT-qPCR and Western blot. The mechanism by which SHP2 knockdown affected the MAPK pathway and its downstream NF-κB pathway under inflammatory conditions was assessed by Western blot. Results: Green fluorescence was observed after transfection for 72 h, and the titer of SHP2 shRNA recombinant lentivirus was 2.9×108 TU/mL. SHP2 expression was significantly downregulated in lentivirus-transfected cells, as demonstrated by Western blot and RT-qPCR (P<0.001). SHP2 knockdown inhibited hPDLSC proliferation to a certain extent and increased the expression of early osteogenic markers under normal conditions, including increased ALP activity and increased ALP and COL-1 expression (P<0.05). However, SHP2 knockdown exerted no effect on mineralized nodule formation. In the TNF-α- and IL-1β-induced inflammatory environment, SHP2 knockdown exerted no effect on hPDLSC proliferation (P>0.05). Osteogenic markers were upregulated (P<0.05), and mineralized nodules were significantly increased (P<0.05) after SHP2 knockdown. Western blot analysis showed that p65 phosphorylation and IκB-α degradation were reduced in SHP2-knockdown hPDLSCs in the inflammatory environment. Moreover, SHP2 knockdown significantly inhibited the expression of p-p38 and p-JNK MAPK, which represent pathways upstream of the NF-κB pathway (P<0.05). Conclusion SHP2 knockdown did not affect cell viability but promoted the osteogenic potential of hPDLSCs by inhibiting the MAPK/NF-κB-mediated signaling pathway under inflammatory environment.