Functional Analysis Of Nitric Oxide Synthase In The BH₄-deficient Silkworms

Tetrahydrobiopterin(tetrahydrobiopterin,BH4)is an essential coenzyme for nitric oxide synthase(nitric oxide synthase,NOS).NOS is a key enzyme in the production of nitric oxide(NO),which is an important cellular signaling molecule.BH4 plays a key role in many biological processes.Sepiapterin reductase(SPR)is a key enzyme in the de novo synthesis pathway of BH4.In order to improve the diagnosis and treatment efficiency of human BH4 deficiency caused by SPR mutations,a large number of studies have been carried out in different model animals.Our previous studies have shown that,in the silkworm Bombyx mori,genetic mutation of sepiapterin reductase(SPR)caused activity partial or total loss is responsible for the lemon mutant phenotypes(leml)and its allelic phenotypes(leml,died after the second instar).In order to understand whether NOS/NO levels and their regulated oxidative stress responses are involved in the lethal process of leml silkworm,BH4-deficient silkworm strains ah09(lem)and a65(leml),wild-type silkworm p50 and silkworm BmN cell lines were used as experimental materials to carry out related research in this paper,and the following research results were obtained.Two NOS genes,BmNOSl and BmNOS2,exist in the silkworm genome.The analysis showed that the silkworm mainly expressed BmNOSl.The results of comparative studies on ah09 and p50 showed that the transcription level of BmNOS1 in the head,silk gland,gonad and trachea of ah09 five-day larvae was significantly lower than that of p50,and in the head,epidermis,midgut,The activities of NOS in silk gland and fat body were lower than p50,indicating that a certain degree of BH4 deficiency affects the expression level of NOS in several tissues of silkworm.Based on the comparison of a65 and p50,it was found that in the a65 of severely deficient BH4,the transcriptional level,enzyme activity level and NO concentration of BmNOS1 were significantly lower than that of wild type,while the content of superoxide anion was significantly higher than that of wild type.When the expression of BmSpr was knocked down in BmN cells,similar results were repeatedly obtained.In order to further identify the role of NOS in the changes of such physiological indicators,we incubated the BmN cells with the NOS inhibitor L-NAME and investigated again,which was found to significantly reduce the decrease of NO and the increase of superoxide anion in the cells,which at a result,directly facilitated the decline of cell proliferation ability.In addition,several antibacterial peptide genes,Attacin2,Cecropin B6,Cecropin D and Defensin,were down-regulated in BmN cells incubated with BH4-deficient silkworm a65(leml)RNAi-BmSpr and NOS inhibitor L-NAME,indicating that the decrease of NO content caused by insufficient synthesis of BH4 can affect the immune function of silkworm.Our studies in vitro and in vivo studies confirm that BmNOS1 has important physiological functions in silkworms.Due to the serious lack of BH4,the leml mutant leads to the uncoupling of NOS,which causes a series of oxidative stress reactions downstream,which is highly likely to cause lethal toxicity to the cells and organisms of leml larvae.