An Induced Disease Model Of Hyperproteinemia Based On Invertebrates And Its Pathological Impact

Hyperproteinemia is a major metabolic condition characterized by abnormally high protein content in the circulatory system.It is commonly seen in cirrhosis,multiple myeloma,angiosarcoma,metabolic acidosis,nephropathy,peritonitis,chronic lymph flow,abdominal abscess,nematode infection,and other diseases and complications.As it is difficult to distinguish the influence of hyperproteinemia and primary disease or infection,on the body in clinical practice,existing reports are limited to compare the differences in the influences of basic metabolic data,such as hyperproteinemia and hypoalbuminemia,and hyperglycemia and hyperlipidemia on blood physical and chemical properties;there is a paucity of relevant research on the pathological mechanism of hyperproteinemia.Thus far,there is no reliable animal model or modeling method of hyperproteinemia.Research on pathogenesis,clinical treatment,and development of therapeutic drugs for hyperproteinemia has been stagnant.Therefore,there is an urgent unmet need to develop a reliable animal model and modeling method of hyperproteinemia.To address this lacuna and on the basis of existing research in our laboratory,we have reconstructed the animal model of hyperproteinemia by using the characteristic Chinese invertebrate model silkworm,investigated the effect of high plasma protein concentration(PPC)on blood metabolism and innate immunity,evaluated the damage and mechanism of influence of high PPC on metabolism and detoxification tissue fat body remodeling and development,and further studied the mechanism of regulation of high PPC reproductive toxicity and its long-term effects on the organism.The main results are as follows:(1)Reconstruction of hyperproteinemia disease modelBased on existing work in our research laboratory,by blocking the orifice of spinneret of mature silkworm larvae,thereby preventing the silk protein in the silk gland from secreting in vitro,and further using the degradation and dissociation process of the silk gland during metamorphosis and development,a large amount of silk protein in the silk gland was rapidly released into the circulatory system.This resulted in a rapid increase in plasma protein concentration(PPC),which was maintained at a level that was 7-fold higher than that of the control silkworm.Hence,a disease model of silkworm hyperproteinemia with different degrees of mortality,controlled from 0% to 100% was constructed.High PPC resulted in abnormal pupation and metamorphosis during metamorphosis and development of the silkworm,and all models with severe hyperproteinemia died.Blood biochemical analysis showed that PPC of the disease model group increased gradually;however,no significant changes in blood glucose,serum triglyceride,or serum total cholesterol were observed.We can,therefore,posit that the silkworm model of hyperproteinemia(AM)with no primary disease and controllable PPC level has been successfully reconstructed.(2)Effect of high PPC on hemolymph metabolismThe results of hemolymph metabolomics analysis showed that the metabolites that were significantly affected due to by high PPC were mainly enriched in amino acid metabolic pathways,which included alanine,aspartic acid,and glutamic acid metabolism,arginine and proline metabolism,D-glutamine and D-glutamic acid metabolism,as well as the metabolism of glycine,serine,and threonine.Metabolic pathway integration analysis showed that metabolic pathways,such as glycolytic pathway,lipid metabolism,TCA cycle,and urea cycle also changed significantly.Among these metabolic pathways,the overall metabolism of amino acids was up-regulated.Several important amino acids,such as tryptophan,phenylalanine,alanine,arginine,threonine,and glutamine were significantly increased.Glucose metabolism was up-regulated,but lipid metabolism and the TCA cycle were down-regulated;the urea content produced by the urea cycle was also significantly reduced.Thus,we showed that high PPC induces amino acid metabolism,but reduces the overall metabolic activity of the body.(3)Effect of high PPC on innate immunity of silkworm blood systemThe results of transcriptome immunoassay of metabolism and detoxification tissue fat body(FB)and GO and KEGG analyses revealed that high PPC results in significant changes in the transcription level of antimicrobial peptides(AMPs)involved in innate immune response and their regulation of the related NF-?B signaling pathway.Further plasma antibacterial activity test results showed that high PPC induced an increase in antibacterial activity of hemolymph against E.coli,but inhibited its antibacterial activity against S.aureus.The m RNA levels of 6 types of AMP genes induced by high PPC in hemocytes and fat bodies were significantly increased,and the m RNA levels of defensin gene in hemocytes was increased by 2300-fold compared to the control(CK)group at 192 h after modeling.The up-regulated expression of AMPs was regulated by the Toll and Imd pathways in NF-?B signaling.High PPC resulted in significant changes in the types of circulating blood cells in hemolymph;the proportion of granulocytes with immune function in the total number of hemocytes increased by about one-fold.High PPC also inhibited the activity of prophenoloxidase(PO)by reducing the m RNA expression of PPO1 and PPO2,thereby reducing the immune effect of hemolymph due to melanin.(4)Effects of high PPC on metabolic tissues fat body remodelingTaking metabolism and detoxification tissue fat body as the object,and the morphological and functional remodeling development of FB during pupation metamorphosis as the target,we showed that high PPC hindered the regeneration and development process of FB,and the remodeling process was delayed for more than 48 h.Pathogenesis studies showed that high PPC decreased autophagy and apoptosis of FB tissue by weakening the effect of endocrine hormones,and prevented the remodeling and regeneration of FB during metamorphic development;meanwhile,high PPC induced increase in oxidative stress and disorder of amino acid transformation function in FB.After modeling,supplementation of 20-hydroxyecdysone,the active substance of ecdysone,can effectively save the delayed regeneration process of FB induced by high PPC.Moreover,the m RNA levels of ecdysone signaling pathway-related genes,Bm Ec R and Bm E74 A,tissue dissociation-related enzyme gene,Bm Cat B,and cell autophagy and apoptosis signaling pathway related genes,Bm Atg6,Bm Atg8,and Bm Dronc all recovered to some extent.(5)Effect of high PPC on reproductive developmentA survey of the far-reaching effects of high PPC on the reproductive development of silkworm showed that high PPC significantly reduced the number and quality of female eggs produced by the silkworm.Further development of gonads was also affected by high PPC,which inhibited the development of male testes and female ovaries and decreased gonads coefficient and delayed development.At the same time,the results of in vitro culture of seminal vesicles showed that after the male gonads had experienced a high-protein hemolymph environment,even after leaving the high PPC environment,there was a significant persistent adverse effect on the development of spermatogenic vesicles in the later period,with toxic accumulation and development of toxicity hysteresis in the silkworm gonads.The results of pathogenic mechanism showed that high PPC inhibited the synthesis of vitellogenin(Vg)in FB,and inhibited the transport of Vg by reducing the signaling pathways of 20-hydroxyecdyone receptors,Ec R and E74.At the same time,high PPC induced autophagy and apoptosis in female ovarian tissues,reduced the expression of Vg receptor Vg R,thereby hindering the absorption of Vg,and,thus,affecting development of the ovary.