Zonghao Tang

Chitosan/kaolin composite porous microspheres with high hemostatic efficacy

The hemostatic performance of chitosan was greatly improved by blending it with kaolin to fabricate porous composite microspheres (CSMS-K) through inverse emulsion method combining with thermally induced phase separation. The CSMS-K had high amount of interior and surface pores. The synergetic hemostatic competence of chitosan and kaolin components made the hemostatic efficacy of CSMS-K superior to chitosan porous microspheres (CSMS). The hemostatic time of CSMS-K3 in the rat tail amputation and liver laceration models was down to respective 120 and 99s from 183 and 134s of CSMS, and the blood loss of CSMS-K3 was respectively 65% and 36% of that of CSMS in the rat tail amputation and liver laceration models. The whole blood clotting kinetics proved that CSMS-K3 formed larger blood clots than CSMS and Celox within a same time period. Our results suggested that the CSMS-K is a potential quick pro-coagulant agent for traumatic hemorrhaging control.

Porous chitosan microspheres for application as quick in vitro and in vivo hemostat

Controlling massive hemorrhage is of great importance to lower transfusional medical cost, and to reduce death and mobility rate in battlefield and civilian accidents. We reported the fabrication of porous chitosan microspheres (CSMS) with tunable surface pore size by microemulsion combined with thermally induced phase separation technique, and its application as a quick hemostat. Their hemostatic property was characterized by blood clotting kinetics, adherence interaction between red blood cells/platelets and CSMS, in vitro and in vivo hemostasis by rat tail amputation and liver laceration models, and histological analysis. Their density, surface area, porosity, water absorption ratio were 0.04-0.06g/cm3, 28.2-31.5m2/g, 98%, and 15.5-23.2g/g, respectively. The surface pore was controlled to be smaller than 2.0μm. The porous CSMS showed increasing hemostatic efficacy with decreasing surface pore size. Compared to the conventional compact chitosan particles (CCSP), the porous CSMS had much improved in vitro and in vivo hemostatic potential with respect to formation of blood clot, hemostatic time, and blood loss. For instance, the hemostatic time and blood loss of CSMS in the rat liver laceration model were down to respectively 70s and 0.026g from 175s and 0.28g of CCSP. Histological examination showed that application of porous CSMS to liver laceration caused no destruction of underlying hepatocytes, inflammatory reaction, and thermal injury to liver tissue. The porous CSMS is a biodegradable, quick and safe hemostat, which can be used in various wounds including complex and non-compressive ones.

Overexpression of hypoxia-inducible factor prolyl hydoxylase-2 attenuates hypoxia-induced vascular endothelial growth factor expression in luteal cells

Vascular endothelial growth factor (VEGF)-dependent angiogenesis has a crucial role in the corpus luteum formation and their functional maintenances in mammalian ovaries. A previous study by our group reported that activation of hypoxia‑inducible factor (HIF)‑1α signaling contributes to the regulation of VEGF expression in the luteal cells (LCs) in response to hypoxia and human chorionic gonadotropin. The present study was designed to test the hypothesis that HIF prolyl‑hydroxylases (PHDs) are expressed in LCs and overexpression of PHD2 attenuates the expression of VEGF induced by hypoxia in LCs. PHD2-overexpressing plasmid was transfected into LC2 cells, and successful plasmid transfection and expression was confirmed by reverse transcription quantitative polymerase chain reaction and western blot analysis. In addition, the present study investigated changes of HIF‑1α and VEGF expression after incubation under hypoxic conditions and PHD2 transfection. PHD2 expression was significantly higher expressed than the other two PHD isoforms, indicating its major role in LCs. Moreover, a significant increase of VEGF mRNA expression was identified after incubation under hypoxic conditions, which was, however, attenuated by PHD2 overexpression in LCs. Further analysis also indicated that this hypoxia‑induced increase in the mRNA expression of VEGF was consistent with increases in the protein levels of HIF‑1α, which is regulated by PHD-mediated degradation. In conclusion, the results of the present study indicated that PHD2 is the main PHD expressed in LCs and hypoxia‑induced VEGF expression can be attenuated by PHD2 overexpression through HIF‑1α‑mediated mechanisms in LCs. This PHD2-mediated transcriptional activation may be one of the mechanisms regulating VEGF expression in LCs during mammalian corpus luteum development.

Porous chitosan microspheres containing zinc ion for enhanced thrombosis and hemostasis

Quick hemostats for non-lethal massive traumatic bleeding in battlefield and civilian accidents are important for reducing mortality and medical costs. Chitosan (CS) has been widely used as a clinic hemostat. To enhance its hemostatic efficiency, Zn2+ in the form of zinc alginate (ZnAlg) was introduced to CS to make porous CS@ZnAlg microspheres with ZnAlg component on the surface. Such microspheres were prepared by successive steps of micro-emulsion, polyelectrolyte adhesion, and thermally induced phase separation. Their structure and hemostatic performance were analyzed by SEM, FT-IR, XPS and a series of in vitro hemostatic experiments including thromboelastography analysis. The composite microspheres had an outer and internal interconnected porous structure. Their size, surface area, and water absorption ratio were ca. 70μm, 48m2/g, and 1850%, respectively. Compared to the neat chitosan microspheres, the CS@ZnAlg microspheres showed shorter onset of clot formation, much faster in vitro and in vivo whole blood clotting, bigger clot, less blood loss, and shorter hemostatic time in the rat liver laceration and tail amputation models. The synergetic hemostatic effects from (1) the electrostatic attraction between chitosan component and red blood cells, (2) the activation of coagulation factor XII by Zn2+ of zinc alginate component, and (3) physical blocking by microsphere matrix, contributed to the enhanced hemostatic performance of CS@ZnAlg microspheres.

Effects of dimethyl carbonate‑induced autophagic activation on follicular development in the mouse ovary

Dimethyl carbonate (DMC) is a widely used industrial chemical, which may be increasingly used in the future. However, its toxicity profile remains largely unknown. The present study was designed to investigate the effects of DMC exposure on the ovaries and the effect of autophagy activation on follicular development. Rats were randomly divided into a control group and low, medium and high dose DMC groups (all n=10). Histological analyses identified no marked differences in the rate of apoptosis between the control and low dose groups; however, marked apoptosis occurred in the medium and high dose groups. The expression of cleaved caspase-3 was significantly increased in the medium and high dose groups, which was consistent with changes observed in the expression of Bcl-2 and Bax. These results indicated that DMC exposure induces toxicity on ovarian function via the induction of apoptosis. The increased expression of the autophagy-related proteins light chain 3II, beclin-1 and p62 following exposure to DMC further indicated that autophagy was activated primarily in the granulosa cells of ovarian follicles in a dose-dependent manner. In addition, the changes in the expression of hypoxia inducible factor 1 α subunit (HIF-1α) and its target protein BCL2 interacting protein 3 (BNIP3) indicated that they may serve a role in the follicular development process induced by DMC. The results of the current study demonstrated that DMC exposure activated autophagy in the ovarian tissue. Furthermore, exposure to low doses of DMC may protect follicular development by activating the HIF-1α/BNIP3 signaling pathway. Taken together, these results indicate that exposure to medium and high doses of DMC induced follicular atresia by activating the apoptotic signaling pathway. This may be an important mechanism of regulating follicular development and ovarian function in mammals.

HIF Prolyl Hydroylase is an Important Element of Biological Information Network on Cell Engneering of the Follicular Growth and Development in Sprague-Dawley Rats

Follicular cell engineering plays an important role in biological engneering and is regulated by different sigaling. The diverse physiological and pathological processes of the ovary are regulated by hypoxia-inducible factor (HIF)-1alpha, a transcription factor, regulated by HIF prolyl hydroylase (PHD) mediated degradation. For further understanding the regulation of HIF-1alpha signaling and the functions of PHDs in ovarian regulating network, the present study firstly developed ovarian follicle development model of postnatal female rats, and then investigated the expression and localization of three PHD is enzymes, PHD1, PHD2 and PHD3 in each ovary through immunohistochemistry. The present results showed that in the ovary all PHD enzymes expressed in a cell-specific manner and changed in a stage-specific manner in vivo. Further observation by real-time PCR found that PHD2 was mainly expressed isoenzyme in the ovary. Together, our present study indicated PHDs may be directly involved in the regulation of ovarian physiological functions through HIF-1alpha signaling, indicating PHD2 is an important element of biological information network on cell engneering of the follicular growth and development in mammals.

Effects of Echinomycin on Follicular Development in the Ovary of Postnatal Rats

Echinomycin (Ech) is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in the regulation of ovarian functions in mammals. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated proliferation cell nuclear antigen (PCNA) expressions contributed to the follicular development in the ovary of postnatal rats. Through the histological examination, the decrease of growing and antral follicle numbers was found in Ech-treated group. And then PCNA mRNA and protein expressions were detected by real time PCR and western blot and the significant decreases of these were found. While the levels of HIF-1alpha mRNA and protein expression were no obviously changes. Further analysis of HIF-1 binding activity found Ech significantly inhibited HIF-1 binding activities in Ech-treated group. These results indicated Ech inhibites follicular development though HIF-1alpha signaling pathway and HIF-1alpha plays an important role in this developmental process in PCNA-dependent manner. Granulosa cell culture experiment further identified the above findings. Taken together, these results demonstrated this HIF-1alpha-mediated PCNA expression is one of the important mechanisms regulating the ovarian follicular development in mammals.