| Heme oxygenase(HO-1)is a key enzyme that catalyzes the breakdown of heme into bilirubin,carbon monoxide and ferrous ions.Most iron in animal bodies are derived from aging red blood cells,in which iron produced by the decomposition of heme.HO-1 plays an important role in iron metabolism.In recent years,related researches have focused on its antioxidant function,but the real role of HO-1 in the body’s iron metabolism is unclear yet.Macrophages,as the main site for HO-1 to break down heme,have both immune function and iron regulation.It is still unclear if the crosstalk of both functions is mediated by HO-1.This study the distribution of HO-1 in important main organs for iron metabolism and figured out the effect of HO-1 on the body’s iron metabolism.Then we explored the effect of HO-1 on the inflammatory response and macrophage functions in mice.The results would provide new ideas for the prevention of iron deficiency in piglets under infected conditions.Test 1:HO-1 expression and cell localization in miceIn this experiment,we explored the expression pattern of HO-1 in iron-absorbed tissues(duodenum and jejunum),iron-storage tissues(liver),iron-recovered tissues(spleen),and kidneys in mice.Real-time PCR results showed that the spleen had the highest HO-1 mRNA expression level,which was significantly higher than that of other tested tissues(p<0.01).The protein expression of HO-1 was similar to its mRNA expression.HO-1 protein was highly expressed in the spleen,but it was almost undetectable in the duodenum.Cell localization results showed that HO-1 was highly coincident with mouse macrophage marker F4/80,indicating that HO-1 was mainly expressed in macrophages.The above results indicate that HO-1 is highly expressed in the spleen of mice and is mainly expressed in macrophages.Test 2:Effect of HO-1 on iron metabolism in mice and macrophagesIn this experiment,a mouse model of HO-1 overexpression and inhibition was constructed by intraperitoneal injection of hemin and ZnPP,respectively,and the effects of HO-1 on iron deposition and gene expression related to iron metabolism were explored.The test results showed that intraperitoneal injection of hemin(30 mg/kg)could significantly increase the expression of HO-1 protein in the spleen,kidney,liver and duodenum of mice,by up to 1.3-folds,while injection of ZnPP(25 mg/kg)significantly reduced the expression of HO-1 protein in the spleen,kidney,and duodenum,which could decrease up to 82.7%,indicating that the HO-1 overexpression and inhibition model was successfully constructed.The body’s iron metabolism results showed:overexpression of HO-1 could significantly increase iron deposition in the spleen tissue,significantly increase the mRNA expression level of ferritin(FtL)in the duodenum,spleen and liver,which increased by 2.7-folds in liver tissue;Overexpression of HO-1 can significantly increase the mRNA expression level of the F erroportin(FPN)in the spleen and liver,which can increase up to 84.3%,meanwhile,overexpression of HO-1 significantly reduces the expression level of divalent metal transporter(DMT1)gene in duodenum by 43.2%.Inhibition of HO-1 significantly reduced the expression level of FPN mRNA in the spleen and liver(p<0.05),which was reduced by 48.2%in the liver.Macrophage RAW264.7 was used for in vitro experiments,and the models of inducing and inhibiting macrophage HO-1 were constructed by adding hemin(30μM)and ZnPP(25 μM)to the culture medium,respectively.The results of iron metabolism in macrophages showed that the induction of HO-1 could increase the intracellular iron concentration of macrophages and significantly increase the mRNA expression levels of ferritin(FtL and FtH).And significantly increased the mRNA expression level of FPN by 83.7-folds,but reduced the mRNA expression levels of iron regulating-related genes Hamp and transferrin receptor(TfRC)by 53.5%and 23.7%,respectively.In contrast,inhibition of HO-1 significantly reduced the gene expression of ferritin(p<0.05),and significantly increased the mRNA expression levels of Hamp and TfRC(p<0.01).The above results showed that HO-1 can significantly affect the iron metabolism of the body and macrophages,that is,increase the body’s iron deposition and increase iron storage,meanwhile slow down the extracellular iron uptake of macrophages iron uptake and accelerate the intracellular iron excretion.Test 3:Effect of HO-1 on inflammatory response and macrophage function in miceIron metabolism is closely related to the function of macrophages.We then explored the effect of HO-1 on the inflammatory response and macrophage function in mice.Based on the mouse model of HO-1 overexpression and inhibition,this experiment constructed an infected mice model by intragastric administration with E.coli K88(2×108 CFU).Then we analyzed inflammatory factors in the duodenum and spleen and the effects of HO-1 on the secretion,polarization,migration and chemotaxis of macrophages were detected in vitro.The results showed that overexpression of HO-1 could significantly increase the mRNA expression of anti-inflammatory factor IL-10 in duodenum and spleen,and significantly reduced the mRNA expression of proinflammatory factor IL-1β by 43.4%.The inhibition of HO-1 significantly increased the mRNA expression of proinflammatory factor IL-6 in duodenum and spleen.Among them,duodenum increased by 1.8-folds,and proinflammatory factor IL-1β in spleen increased by 35.1%.Cell test results showed that overexpression of HO-1 significantly reduced IL-1β mRNA expression by 56.9%,significantly reduced iNOS mRNA expression by 19.6%,and significantly increased IL-10 mRNA expression by 1.3-folds;while inhibition of HO-1 significantly increased IL-6 mRNA expression by 3.0-folds.In summary,HO-1 can inhibit the inflammatory response,significantly inhibit the expression of pro-inflammatory factors IL-1β and IL-6,and promote the expression of anti-inflammatory factor IL-10.Macrophage function test results show that overexpression of HO-1 can significantly reduce the protein expression level of M1-type polarization marker factor iNOS and the mRNA expression levels of iNOS,IL-6 and Cox2,of which iNOS could be reduced by 73.0%,which could significantly increase M2 polarized markers Arg-1,IL-10 and Fizzl mRNA expression levels(p<0.001);overexpression of HO-1 could significantly increase macrophage migration rate(p<0.01),significantly increase MCP-1,mRNA expression levels of chemokines such as CCL8.Inhibition of HO-1 expression showed completely opposite results.The above results show that HO-1 can inhibit the body’s inflammatory response,induce macrophages to polarize to M2-type,increase the migration rate of macrophages,and promote the expression of macrophage chemokines.Test 3:Effect of HO-1 on inflammatory response and macrophage function in miceTo evaluate the effect of HO-1 on the inflammatory response and macrophage function relating to iron metabolism,we performed in vitro and in vivo experiments.Based on the mouse model of HO-1 overexpression and inhibition,an infected mice model was built by intragastric administration with E.coli K88(2×108 CFU).Then inflammatory factors in the duodenum and spleen were analyzed,and the effects of HO-1 on the secretion,polarization,migration and chemotaxis of macrophages were detected in vitro.The results showed that overexpression of HO-1 could significantly increase the mRNA expression of anti-inflammatory factor IL-10 in duodenum and spleen,and markedly reduced the mRNA expression of proinflammatory factor IL-1βby 43.4%.The inhibition of HO-1 significantly increased the mRNA expression of proinflammatory factor IL-6 in duodenum and spleen which was increased by 1.8-folds in duodenum.Cell test results showed that overexpression of HO-1 remarkably reduced IL-1β and iNOS mRNA expression by 56.9%and 19.6%respectively but significantly increased IL-10 mRNA expression by 1.3-folds.Meanwhile inhibition of HO-1 markedly increased IL-6 mRNA expression by 3.0 times.In summary,HO-1 can inhibit the inflammatory response,significantly inhibit the mRNA expression of pro-inflammatory factors IL-1β and IL-6,and promote the mRNA expression of anti-inflammatory factor IL-10.Macrophage function test results show that overexpression of HO-1 can significantly reduce the protein expression level of Ml-type polarization marker factor iNOS and the mRNA expression levels of iNOS,IL-6 and Cox2,of which iNOS could be reduced by 73.0%,which could significantly increase M2 polarized markers Arg-1,IL-10 and Fizzl mRNA expression levels(p<0.001);overexpression of HO-1 can significantly increase macrophage migration rate(p<0.01),significantly increase MCP-1 mRNA expression levels of chemokines such as CCL8.The inhibition of HO-1 expression showed completely opposite results.The above results show that HO-1 can inhibit the body’s inflammatory response,induce macrophages to polarize to M2 type,increase the migration rate of macrophages,and promote the expression of macrophage chemokines.In summary,HO-1 can increase the body’s iron deposition and increase iron storage,while slowing down macrophages’ extracellular iron uptake and accelerating intracellular iron excretion;it can inhibit the body’s inflammatory response and induce macrophages to M2-type polarization promotes the expression of chemokines in macrophages and increases the migration rate of macrophages.This study shows that HO-1 can regulate the body’s iron metabolism and has an important effect on the function of macrophages.It may play a certain role in the body’s immunity,providing a certain theory for the prevention and control of iron deficiency in piglets under infected conditions. |
