In situ RT-PCR detection of inducible nitric oxide synthetase gene expression in lung during endotoxemia in rabbits

To detect the location of inducible nitric oxide synthetase (iNOS) protein and mRNA in lung during endotoxemia in rabbits Methods Northern blotting was performed before, 1 hour and 5 hours after the intravenous administration of lipopolysaccharide (LPS) in rabbits Immuno^histochemical analysis (IA), in situ hybridization and in situ reverse transcription polymerase chain reaction (in situ RT PCR) were also performed in lung sections Results iNOS mRNA expression was found using Northern blotting in lung 5 hours after LPS injection, while it was not found in control The positive stain was found only in macrophages in lung 5 hours after LPS injection by standard hybridization and IA; while by in situ RT PCR, the amplification products were found in macrophages, airway epithelial cells, vascular endothelial cells, smooth muscle cells and leukocytes, in addition to macrophages distributed abundantly throughout the lung The signal was absent in control or samples Conclusions Using an in situ RT PCR technique, iNOS expression was not only observed in macrophages but also in many other kinds of cells in lung during endotoxemia in rabbits This suggests that in situ RT PCR is much more sensitive than in situ hybridization, and can be used to examine genes with low expression

Effect of Purified Xuefu Capsule (精制血府胶囊)on Endothelin, Nitric Oxide Synthetase Gene Expression of Vascular Wall in Atherosclerotic Rabbits

Objective: To observe the effect of purified Xuefu Capsule （PXC） on endothelin （ET） and nitric oxide synthetase （NOS） gene expression and proliferation of vascular smooth muscle cell （VSMC） in atherosclerotic rabbits. Methods: Molecular biological techniques of dot blot and in situ hybridization were adopted. Results: The expression of ET mRNA in atherosclerosis （AS）, plaques of the model group was higher,with the positive signals distributed mainly in arterial intimal AS plaques; while at the same time the expression in the PXC treated group was lower, with only few scattering signals found in arterial intimal AS plaques.NOS mRNA expression was less in the vascular wall of the AS model group, PXC could enhance NOS mRNA expression, positive signals could be found from intima to media. Conclusion: The effect of PXC on preventing and treating AS might be related to regulating the expression of ET mRNA and NOS mRNA in the vascular wall.

Specific generation of nitric oxide in mitochondria of cancer cell for selective oncotherapy

Nitric oxide(NO)gas therapy,especially,L-arginine(L-Arg)-based NO treatment strategies have attracted extensive attention in the field of oncotherapy.However,current strategies are unable to differentiate well between normal cells and cancer cells,which may lead to unpredictable toxicity.Motivated by the fact that mitochondria of cancer cells can express excessive nitric oxide synthetase(NOS),herein,a nanozyme-based NO generator,cerium oxide(CeO_(2))-AT,is fabricated to specifically catalyze the production of NO in cancer cells for selective tumor treatment.In this system,after being endocytosed into cancer cells,the generator can produce a number of NO under the catalysis of NOS in mitochondria of cancer cells,which can disrupt the mitochondrial respiratory chain of tumor cells and further induce cell apoptosis.In addition,the generator with catalase(CAT)-like activity can catalyze H_(2)O_(2)to produce O_(2),which can promote the generation of NO and improve the performance of NO gas therapy.What is more,our system has no obvious impact on the viability of normal cells owing to the less production of NO.Our work paves a new way for the development of highly selective NO-based treatment particularly useful for the safe and specific cancer therapy.

Radix Ilicis Pubescentis total flavonoids ameliorates neuronal damage and reduces lesion extent in a mouse model of transient ischemic attack

Flavonoids are a major component in the traditional Chinese medicine Radix Ilicis Pubescentis.Previous studies have shown that the administration of Radix Ilicis Pubescentis total flavonoids is protective in cerebral ischemia.However,to our knowledge,no studies have examined whether the total flavonoids extracted from Radix Ilicis Pubescentis prevent or ameliorate neuronal damage following transient ischemic attacks.Therefore,Radix Ilicis Pubescentis total flavonoids question and the potential underlying mechanisms.Thus,beginning 3 days before the induction of a mouse model of transient ischemic attack using tert-butyl hydroperoxide injections,mice were intragastrically administered 0.3,0.15,or 0.075 g/kg of Radix Ilicis Pubescentis total flavonoids daily for 10 days.The results of spectrophotometric analyses demonstrated that Radix Ilicis Pubescentis total flavonoids enhanced oxygen free radical scavenging and reduced pathological alterations in the brain.Hematoxylin-eosin staining results showed that Radix Ilicis Pubescentis total flavonoids reduced hippocampal neuronal damage and cerebral vascular injury in this mouse model of transient ischemic attack.These results suggest that the antioxidant effects of Radix Ilicis Pubescentis total flavonoids alleviate the damage to brain tissue caused by transient ischemic attack.