Neuroprotective effects of combined lead and cadmium,as well as N-acetylcysteine,on cerebral cortical neurons following lipid peroxidation injury

BACKGROUND： Studies have reported the antioxidant effects of lead and cadmium in the central nervous system, but very few have addressed the combined toxicity of lead and cadmium. The mechanisms by which these combined heavy metals are toxic, as well as how to protect cells from these agents, remains poorly understood. OBJECTIVE： Primary cultured rat cortical neurons were used to determine the effects of combined lead and cadmium on levels of glutathione peroxidase （GSH-Px）, superoxide dismutase （SOD）, catalase （CAT）, and acetylcholinesterase （ACHE）, as well as malondialdehyde （MDA）, and to evaluate the neuroprotective effects of N-acetylcysteine （NAC）. DESIGN, TIME AND SETTING An in vitro toxicological observation was performed at the Comparative Medicine Center of Yangzhou University from August 2007 to April 2008. MATERIALS： Lead acetate, cadmium acetate, and NAC were purchased from Sigma-Aldrich （St. Louis, USA）. Commercial kits of GSH-Px, SOD, CAT, ACHE, and MDA were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. METHODS： The cerebral cortical neurons were isolated from newborn Sprague dawley rats at 24 hours after birth and primary cultured for 6 days. Thereafter, the cells were treated with a range of cadmium doses （0, 5.0, and 10.0μmol/L）, lead doses （0, 1.0, and 2.0 μmol/L）, or a combination of the two for 12 hours at 37℃in a 5% CO2 incubator, respectively. In addition, the cells were incubated with different doses of cadmium and/or lead and （0 and 50 μmol/L） NAC for 12 hours to assess the protective effects on cell survival. MAIN OUTCOME MEASURES： The activity of SOD, GSH-Px, CAT, and ACHE, as well as MDA content, in the cell lysates was detected using commercial kits. RESULTS： At 12 hours after treatment, compared to the control group, activity of GSH-Px, SOD, and AChE in the lead, cadmium, or combined treated cells was significantly decreased with increasing doses of cadmium/or lead （P 〈 0.05）, but CAT activity and MDA levels were significantly increased （P 〈 0.05）. The combination of cadmium and lead led to higher levels of toxicity than individual exposure. CONCLUSION： The degree of oxidative damage increased when the two heavy metals were combined. NAC protected neonatal cortical neurons by increasing activity of anti-oxidative enzymes and reducing lipid peroxidation, but the reduction was not statistically significant.

Construction and Identification of Mammary Gland-specific Expression Vector of Bovine Tracheal Antimicrobial Peptide (TAP)

[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and KpnI, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein （ EGFP）, and transfected into bovine mammary epithelial cells （bMEC）, COS-7 cells and lactating rabbit mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue was detected by semi-quantity RT-PCR. [ Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition, semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfeeted with pBLG-EGFP-TAP. [ Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.

Expression of VFA Receptors GPR41 and GPR43 in Cow Abomasums

With the enlargement of intensive feeding scale of dairy cows and improvement of milk yield, the incidence rate of displacement of abomasum in dairy cows increases gradually in recent years. Previous researches have shown that VFA in abomasums can significantly inhibit the shrinkage of smooth muscles of abomasums and lead to atony of abomasums, but the specific regulatory mechanisms are still unclear. In this study, the expression of VFA receptors GPR41 and GPR43 in cow abomasums were detected by PCR method, and the fragments were consistent with the excepted sizes. This study provided theoretical basis for the pathologic mechanism investigation, prevention and treatment of displacement of abomasum in dairy cows.

Zinc alleviates the heat stress of primary cultured hepatocytes of broiler embryos via enhancing the antioxidant ability and attenuating the heat shock responses

Zinc(Zn)has been shown to attenuate the adverse effects of heat stress on broilers,but the mechanisms involving this process remain unclear.We aimed to investigate possible protective mechanisms of Zn on primary cultured hepatocytes of broiler embryos subjected to heat stress.Three experiments were conducted.In Exp.1,hepatocytes were treated with 0,50,100,200,or 400μmol/L added Zn as inorganic Zn sulfate(iZn)for 12,24 or 48 h.In Exp.2,cells were exposed to 40℃(a normal temperature[NT])and 44℃(a high temperature[HT])for 1,2,4,6,or 8 h.In Exp.3,cells were preincubated with 0 or 50μmol/L Zn as iZn or organic Zn lysine chelate(oZn)for 8 h under NT,and then incubated with the same Zn treatments under NT or HT for 4 or 6 h.The biomarkers of antioxidative status and heat stress in cells were measured.The results in Exp.1 indicated that 50μmol/L Zn and 12 h incubation were the optimal conditions for increasing antioxidant ability of hepatocytes.In Exp.2,the 4 or 6 h incubation under HT was effective in inducing heat shock responses of hepatocytes.In Exp.3,HT elevated(P<0.01)malondialdehyde content and expressions of heat shock protein 70(HSP70)mRNA and protein,as well as HSP90 mRNA.However,Zn supplementation increased(P<0.05)copper zinc superoxide dismutase(CuZnSOD)activity and metallothionein mRNA expression,and effectively decreased(P<0.05)the ex-pressions of HSP70 mRNA and protein,as well as HSP90 mRNA.Furthermore,oZn was more effective(P<0.05)than iZn in enhancing CuZnSOD activity of hepatocytes under HT.It was concluded that Zn(especially oZn)could alleviate heat stress of broiler hepatocytes via enhancing their antioxidant ability and attenuating heat shock responses.