Effect Of Huoxuehuayu Decoction On The Enzymatic Activities Of Nitric Oxide Synthase And Inducible Nitric Oxide Synthase Genetic Expression In Rats During Frature Healing

Objective: Observe the effects of Huo Xue Hua Yu(HXHY) decoction on serum enzymatic activities of nitric oxide synthase(NOS) and callus tissue inducible NOS(iNOS) genetic expression during fracture healing in rat at level of enzymatic activity, mRNA and protein, in order to explicate the potential molecular mechanism of HXHY decoction during fracture healing.Methods:Standardized fracture model of the left tibia in 80 isoflurane-anesthetized male SD rats with a weight of 320 ± 15g was induced using a modified technique. Frature sides were fixed by percutaneous intramedullary nails(76 rats up to commamd). All rats were filled with HXHY decoction served as treatment group while distilled water served as control group after fracture. Blood serum was determined by the enzymatic activity of NOS, iNOS and constitutive NOS (cNOS) 1^st before fracture and on 1^st, 4^th, 7^th, 14^th, 21^st after fracture(n=20/time point). Callus of fracture sides were determined by the iNOS mRNA expression, protein expression on 4^th, 7^th, 14^th ,21^st (n=14/time point) via reverse transcriptase polymerase chain reaction (RT-PCR) and western blottings.Results: 1. Serum NOS, cNOS, iNOS enzymatic activities of control group reach peak value on 14^th, 4^th, 14^th after fracture separately, Serum NOS and cNOS enzymatic activities in treatment group reach peak value on 14^th, while iNOS enzymatic activities increased continuously. After treatment of HXHY decoction, the enzymatic activity of NOS keeps higher on 4^th (p<0.05), 7^th, 4^th and21"(p<0. 01)during fracture healing, the enzymatic activity of cNOS increased significantly on 7th(p<0. 05), 14th(p<0. 01), iNOS on 4th and 21 lh(p<0. 05) after fracture. 2. All callus tissue iNOS mRNA expression increased on 7lh, 14 th and 21st with the peak on the 14 lh. There is an significantly increase on iNOS mRNA expression especially on 7 thand 14lh(p<0. 01) and protein expression on 14 th after treatment by HXHY decoction.Conclusions: 1. Standardized percutaneous intramedullary nail fracture model of the tibia in rats using a modified technique fixed have the characters of stability, reliability, reproducibity. 2. The activity increase of serum NOS enzyme, cNOS and iNOS after the fracture changes differently;4dcN0S has the highest activity while iNOS have the minimum activity after the fracture, 14dcN0S has the minimum activity and iNOS have the highest activity after the fracture. 3. HXHY decoction improve the enzymatic activity of NOS, mainly improving iNOS enzymatic activity on 4lh, 21 '\ and cNOS enzymatic activity on 7 th, 14 l\ thus accelerating fracture healing. 4. The results of this experiment indicate that the reason why HXHY decoction improve iNOS enzymatic activity is that HXHY decoction accelerats callus iNOS mRNA expression and protein expression on during early fracture healing.