膳食是人类赖以生存的物质基础，膳食不均衡往往会引起一系列的健康问题。一些流行病学研究认为长期摄入过量的肉类会增加结直肠炎症甚至癌症等的风险。通常专家们认为肉中的血红素是引起这一变化的主要因素，但是我们前期的研究发现肉中的肌红蛋白的消化性差，经胃肠道消化后它是否会释放出血红素还未可知。基于此，本研究将从猪心肌中提取的肌红蛋白，以不同剂量添加到酪蛋白饲料中模拟不同剂量肉类的摄入，根据AIN-93M标准配方配制饲料。6周龄C57BL/6J小鼠适应两周后，自由采食3周或8周，探究短期和长期摄入不同剂量的肌红蛋白对小鼠生理状况、肠道微生物、代谢物和宿主肠道健康的影响及相关的机制，并通过探究饲料消化产物对INT407细胞和HT29细胞的细胞活力、凋亡、周期及氧化状态的影响，以期解释不同含量的肌红蛋白影响肠道健康的原因，为合理膳食指导提供一定的理论依据。具体研究如下：

1. 肌红蛋白对小鼠肠道健康的影响

为了探究不同含量的肌红蛋白和不同饲喂时间对小鼠生理健康的影响。采用硫酸铵沉淀法和超滤洗脱法从猪心肌中提取肌红蛋白，将其添加到饲料中以配制AIN 93M饲料。以C57BL/6J小鼠为对象，饲喂对照饲料（CON，14%酪蛋白）、低剂量肌红蛋白饲料（LMb，0.38%肌红蛋白 + 13.62%酪蛋白）、中剂量肌红蛋白饲料（MMb，1.13%肌红蛋白 + 12.87%酪蛋白）和高剂量肌红蛋白饲料（HMb，3.39%肌红蛋白 + 10.61%酪蛋白），每3天记录采食量和体重。在饲喂3周和8周结束时，测量小肠和结肠的长度和微观结构，并称量小鼠的肝脏、脾脏、附睾脂肪以及盲肠重量，测定血清中内毒素（LPS）、二胺氧化酶（DAO）和D-乳酸的含量。结果显示，高肌红蛋白组小鼠的平均日增重和平均日采食量明显低于其他组。随着肌红蛋白含量的增加，肝脏指数和肾脏指数显著增加，脾脏指数和附睾脂指数降低，盲肠组织及其内容物重量最大和盲肠指数显著增加。在8周结束时，小鼠的结肠长度和小肠长度均是随着肌红蛋白含量的增加而增加。此外，血清中DAO、D-乳酸和LPS的含量也随之增加，在HMb组最高。与CON组相比，MMb和HMb组的十二指肠的隐窝深度增加和绒毛高度降低，肠壁变薄，绒毛间隙变大，炎性细胞浸润，绒毛完整性较差。同时，结肠发生炎性细胞浸润，杯状细胞数量减少、黏液层厚度降低，肠道黏液分泌减弱和肠道屏障受损。这些结果表明肌红蛋白显著影响小鼠的肠道健康，与酪蛋白相比，LMb组无显著差异，而HMb组不利于小鼠的生长，造成肠道通透性增加和肠道组织受损。

2. 肌红蛋白影响肠道稳态的生物机制探究

采集小鼠十二指肠和结肠组织，对肠道黏液和紧密连接蛋白相关基因、氧化应激相关指标、炎症因子含量进行测定。十二指肠的紧密连接蛋白（ZO-1、Occuludin和Claudin 1），黏蛋白（MUC 1和MUC 2）的mRNA表达量在HMb组显著降低（P < 0.05）。随着肌红蛋白的增加，十二指肠的MDA、4-HNE和8-OHdG均显著增加（P < 0.05），SOD活力增加，并且肿瘤坏死因子（TNF-α）和炎症因子（lL-1β和IL-6）也随之增加（P < 0.05）。同样地，结肠的紧密连接蛋白（ZO-1、Occuludin和Claudin 1），黏蛋白（MUC 1和MUC 2）的mRNA表达量在LMb组显著最高，在HMb组显著最低（P < 0.05）。肌红蛋白的增加可以显著增加结肠组织中4-HNE、MDA和8-OHdG的含量，SOD活力降低（P < 0.05）。结肠组织的脂质过氧化程度增加，ROS产生过多，抗氧化能力下降，同时，DNA损伤增加。在3周时，随着肌红蛋白含量增加，TNF-α、IL-6和IL-1β的mRNA表达量增加，在8周时，HMb组炎症因子的mRNA表达量最高（P < 0.05），这一结果表明高肌红蛋白的摄入可能通过血红素的积累改变氧化应激状态，引发炎症反应，影响肠道屏障的正常功能。

3. 肌红蛋白影响肠道稳态的化学基础机制探究

采集小鼠十二指肠和结肠组织、内容物和粪便，对血红素含量、与血红素分解代谢相关物质含量、血红素转运和铁循环相关基因进行测定。结果表明，随着肌红蛋白含量的增加，十二指肠组织中游离血红素的含量显著增加而内容物中血红素显著降低，血红素加氧酶1（HO-1）、胆红素和胆绿素含量也随之显著增加（P < 0.05），预示血红素被不断的分解代谢，同时，溶质载体家族46成员1（SLC46A1）的mRNA表达量随之增加（P < 0.05），更多的血红素被转运到十二指肠组织中。血红素被分解会释放出铁离子，在3周时，随着肌红蛋白含量的增加，肠道组织中的储铁蛋白（Ferritin）和膜铁转运蛋白（FPN）的mRNA表达量显著增加（P < 0.05），在8周时，Ferritin的mRNA表达量显著增加（P < 0.05），FPN在各组间并无显著性差异，可能导致铁外排功能异常。同时，随着肌红蛋白的增加，结肠内容物的血红素含量显著增加，粪便中的血红素却显著降低，结肠组织中的血红素仅在HMb组显著增加（P < 0.05）。在3周时结肠中SLC46A1的mRNA表达量显著增加，HO-1的mRNA表达量先显著增加后降低（P < 0.05），推测HMb组的血红素被SLC46A1更多的转运到组织中，HO-1的血红素分解降低，即更多的血红素被储存在结肠组织中。综上，高肌红蛋白通过增加组织中血红素的含量生成血红素毒性因子，改变体内铁循环的相关基因Ferritin和FPN的mRNA表达量来影响机体铁稳态，引起肠道氧化应激水平的失衡，从而造成肠道的损伤。

4. 肌红蛋白诱导肠道菌群和代谢产物变化

为了进一步探讨肌红蛋白对结肠黏膜屏障产生上述影响的原因，测定肠道微生物群组成、短链脂肪酸及代谢产物含量。研究表明，随着膳食中肌红蛋白含量的增加，在3周时，随着肌红蛋白的增加，Akkermansia, Bifidobacteria的相对丰度显著增加，而Lachnospiraceae NK4A136 group显著减少（P < 0.05），在8周时仅有Akkermansia显著增加（P < 0.05）。此外，在3周时短链脂肪酸的含量随着膳食中肌红蛋白含量的增加呈现先升高后降低的趋势，而在8周时高肌红蛋白组中丙酸盐和异戊酸盐的含量显著增加（P < 0.05）。此外，粪便代谢组学表明，KEGG差异代谢物主要集中在氨基酸代谢（主要色氨酸代谢）、脂质代谢和消化吸收系统（主要蛋白质消化）这三个方面。对KEGG功能通路中与蛋白质消化相关的代谢物分析，发现肌红蛋白组的对甲酚、间甲酚、苯酚、L-色氨酸和L-半胱氨酸的含量增加，可能引起肠道炎症、细胞毒性增加等。对KEGG功能通路中与色氨酸相关的代谢物分析，发现肌红蛋白可以通过改变吲哚及其衍生物的含量，降低犬尿氨酸代谢中的关键物质，如5-羟基-L-色氨酸和6-羟基犬尿喹啉酸，引起肠道炎症、神经发育、肠道内稳态的改变。

5. 肌红蛋白对肠道健康影响的体外验证

将肌红蛋白进行体外消化处理，结果表明，随着肌红蛋白的增加，胃蛋白酶和胰酶的消化率显著降低（P < 0.05），一些大分子蛋白质以聚集或沉淀在底部，粒径变大，Zeta电位值更低。肌红蛋白含有更高的α-螺旋含量，即氢键含量较多，“刚性”结构更强可能是肌红蛋白具有更低的消化率的原因。基于以上，研究了不同浓度膳食消化产物（0.25、0.5、1、2 mg/mL）在不同处理时间下（12和24 h）对INT407细胞和HT29细胞的细胞活力的影响，确定0.25和1 mg/mL两个剂量进行后续细胞周期和细胞凋亡的实验，测定在细胞损伤较大（1 mg/mL）时，细胞内的脂质过氧化产物丙二醛（MDA）和超氧化物歧化酶（SOD）水平。结果表明，高浓度的膳食消化产物处理后造成INT407细胞和HT29细胞的细胞活力下降，HMb组造成的细胞活力降低更显著（P < 0.05）。同时，培养INT407细胞的24 h时的细胞活力要显著低于12 h，而HT29细胞在12和24 h并无显著差异。在0.25和1 mg/mL时，肌红蛋白消化产物处理细胞后增加了细胞凋亡，对HT29细胞的影响要大于INT407细胞，消化产物浓度越高，细胞凋亡越高，尤其是早期凋亡。0.25和1 mg/mL的肌红蛋白消化产物培养INT407细胞时，延长了细胞周期中G2期，缩短了G1和S期。1 mg/mL的肌红蛋白消化产物培养HT29细胞的结果与之相一致。0.25 mg/mL培养HT29细胞时，延长了细胞周期中S期，缩短了G1期。此外，随着肌红蛋白含量的增加，细胞内MDA的生成和积累，SOD活力被显著抑制。以上结果表明细胞培养实验中，高肌红蛋白会造成INT407细胞和HT29细胞发生细胞活力降低、细胞凋亡增加和细胞周期停滞，并增加脂质过氧化和抑制抗氧化酶的活力。综上，高剂量的肌红蛋白对肠道的黏膜屏障造成的损伤，是通过血红素诱发的过度氧化应激反应引起的，而低剂量的肌红蛋白不会引起肠稳态的改变。

Diet is the material basis for human survival, and unbalanced diet often causes a series of health problems. Some epidemiological studies believe that long-term intake of excessive meat could increase the risk of colorectal inflammation and even cancer. Generally， experts think that heme in red meat is the main factor causing this change, but our previous research found that myoglobin in meat had poor digestibility, and whether it would release heme after digestion in the gastrointestinal tract is unknown. Based on this, myoglobin was added to the casein feed at different doses to simulate the intake of meat at different doses, and the feed was prepared according to the standard formula of AIN-93M. Six week old C57BL/6J mice were fed freely for 3 or 8 weeks after two weeks of adaptation, to explore the effects of short-term and long-term intake different doses of myoglobin on the physiological status of mice, gut microbiota, metabolites and host intestinal health and the related mechanisms, and to explore the effects of feed digestion products on the cell viability, apoptosis, cycle and oxidative status of INT407 cells and HT29 cells, It is expected to explain the reasons why different levels of myoglobin affect intestinal health, and provide a theoretical basis for rational dietary guidance. The specific research is as follows:

1. Effect of myoglobin on intestinal health in mice

In order to explore the effects of myoglobin with different content and different feeding time on the physiological health of mice. Myoglobin was extracted from pig myocardium by ammonium sulfate precipitation and ultrafiltration elution, and added to feed to prepare AIN 93M feed. C57BL/6J mice were fed with control diet (CON, 14% casein), low dose myoglobin diet (LMb, 0.38% myoglobin+13.62% casein), medium dose myoglobin diet (MMb, 1.13% myoglobin+12.87% casein) and high dose myoglobin diet (HMb, 3.39% myoglobin+10.61% casein), and the food intake and body weight were recorded every 3 days. At the end of 3 and 8 weeks of feeding, measure the length and observe the microstructure of small intestine and colon, the weight of liver, spleen, epididymal fat and caecum, and determine the content of lipopolysaccharide (LPS), diamine oxidase (DAO) and D-lactic acid in serum. The results showed that the average daily weight gain and average daily food intake of mice in HMb group were significantly lower than those in other groups (P < 0.05). With the increase of myoglobin content, liver index and kidney index increased significantly, while spleen index and epididymal fat index decreased (P < 0.05). The weight of cecum tissue and its contents was the largest and the cecum index also increased significantly (P < 0.05). At the end of 8 weeks, the length of colon and small intestine of mice increased as myoglobin content increased. In addition, the content of LPS, DAO and D-lactic acid in serum also increased, which was the highest in HMb group (P < 0.05). Compared with CON group, the crypt depth of duodenum in MMb and HMb groups increased and the villus height decreased, the intestinal wall became thinner, the villus space became larger, inflammatory cells infiltrated, and the villus integrity was poor. Meanwhile, inflammatory cell infiltration occurred in colon tissue, the number of goblet cells decreased, the thickness of mucus layer decreased, intestinal mucus secretion weakened and intestinal barrier was damaged. These results indicated that myoglobin significantly affects the intestinal health of mice. Compared with casein, LMb group had no significant difference, while HMb group was not conducive to the growth of mice, which may cause increased intestinal permeability and intestinal tissue damage.

2. Study on biological mechanism of myoglobin on intestinal homeostasis

The tissues and content of duodenum and colon in mice were collected to determine the intestinal mucin and tight junction protein related genes, oxidative stress related indicators, and inflammatory factors. The mRNA expression of duodenal tight junction proteins (ZO-1, Occuludin and Claudin 1) and mucin (MUC 1 and MUC 2) decreased significantly in HMb group (P < 0.05). With the increase of myoglobin, MDA, 4-HNE and 8-OHdG in duodenum were significantly increased (P < 0.05), SOD activity was increased, tumor necrosis factor (TNF-α） and inflammatory factors (lL-1β and IL-6) also increased (P < 0.05). Similarly, the mRNA expression levels of tight junction proteins (ZO-1, Occuludin and Claudin 1) and mucins (MUC 1 and MUC 2) in the colon were significantly the highest in LMb group and the lowest in HMb group (P < 0.05). The increase of myoglobin could significantly increase the contents of 4-HNE, MDA and 8-OHdG in colon tissue, and SOD activity decreased (P < 0.05). The lipid peroxidation degree of colon tissue increased, ROS was produced too much, and the antioxidant capacity decreased, in addition, DNA damage increased. At 3 weeks, the mRNA expression of inflammatory factors (TNF-α、IL-6 and IL-1β) increased as myoglobin increased. At 8 weeks, the mRNA expression was highest in the HMb group (P < 0.05). The above results indicated that the intake of high myoglobin could change the oxidative stress state through the accumulation of heme, trigger inflammatory reaction, and affect the normal function of intestinal barrier.

3. Study on the chemical basis of myoglobin affecting intestinal homeostasis

The tissues and content of duodenum and colon, feces in mice were collected to determine the heme content, the content of substances related to heme catabolism, heme transport and iron cycle related genes. The results showed that the content of free heme in duodenal tissue increased significantly as myoglobin content increased, while the content of heme decreased significantly, and the contents of heme oxygenase-1 (HO-1), bilirubin and biliverdin also increased significantly (P < 0.05), indicating that heme was continuously catabolized, moreover, the mRNA expression of 46 member 1 of the solute carrier family (SLC46A1) increased (P < 0.05), more heme is transported to duodenal tissue. When heme is decomposed, iron ions will be released. At 3 weeks, with the increase of myoglobin content, the mRNA expression of ferritin and membrane iron transporter (FPN) in duodenal tissue increased markedly (P < 0.05). At 8 weeks, the mRNA expression of ferritin increased markedly (P < 0.05), FPN had no significant difference among groups, which led to abnormal iron efflux function. Meanwhile, the heme content of colon contents increased significantly as myoglobin increased, but the heme content in feces decreased significantly. The heme content in colon tissue only increased significantly in HMb group (P < 0.05). At 3 weeks, the mRNA expression of SLC46A1 was significantly increased in the colon, and the mRNA expression of HO-1 was significantly increased first and then decreased (P < 0.05). It was speculated that heme in HMb group was more transported to tissues by SLC46A1, and heme decomposition catalyzed by HO-1 is reduced, and more heme is stored in colon tissue. To sum up, high myoglobin can affect the iron homeostasis of the body by increasing the content of heme in the tissues to generate heme toxic factors, and changing the mRNA expression of Ferritin and FPN, which are related to iron circulation in the body, causing imbalance of intestinal oxidative stress level, thus result in intestinal damage.

4. Changes of gut microbiota and metabolites induced by myoglobin

In order to further explore the reasons for the above effects of myoglobin on the colonic mucosal barrier, the composition of gut microbiota, the content of short chain fatty acids (SCFAs) and metabolites were determined. The research showed that the relative abundance of Akkermania muciniphila and Bifidobacteria increased significantly as myoglobin increased at 3 weeks, while that of Lachnospiracea NK4A136 group decreased significantly (P < 0.05), and only Akkermania muciniphila increased significantly at 8 weeks ((P < 0.05). In addition, the content of SCFAs increased first and then decreased as myoglobin increased at 3 weeks, while the content of propionate and isovalerate in HMb group increased significantly at 8 weeks (P < 0.05). In addition, fecal metabolomics showed that KEGG differential metabolites were mainly concentrated in amino acid metabolism (mainly tryptophan metabolism), lipid metabolism and digestion and absorption system (mainly protein digestion). The analysis of metabolites related to protein digestion in KEGG functional pathway showed that the content of P-cresol, M-cresol, phenol, L-tryptophan and L-cysteine in myoglobin group increased, which may cause intestinal inflammation, increased cytotoxicity, etc. Analysis of tryptophan related metabolites in the KEGG functional pathway showed that myoglobin could reduce key substances in the metabolism of kynurenine, such as 5-hydroxy-L-tryptophan and 6-hydroxycaninurolinic acid, by changing the content of indole and its derivatives, and may induce intestinal inflammation, neural development, and changed in intestinal homeostasis.

5. In vitro validation of the effect of myoglobin on intestinal health

Myoglobin was digested in vitro, the results showed that with the increase of myoglobin, the digestibility of pepsin and pancreatin decreased significantly (P < 0.05). Some macromolecular proteins were aggregated or precipitated at the bottom, the particle size became larger, and the Zeta potential value was lower. Myoglobin contains higher α- helix content, that is, more hydrogen bonds and stronger "rigid" structure, may be the reason why myoglobin has lower digestibility. Based on the above, the effects of different concentrations of dietary digestive products (0.25, 0.5, 1, 2 mg/mL) on the cell viability of INT407 cells and HT29 cells were studied at different treatment times (12 and 24 h). Two doses of 0.25 and 1 mg/mL were determined for subsequent cell cycle and apoptosis experiments. Furthermore, when the cells were damaged greatly (1mg/mL), the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in cells were measured. These results showed that the cell viability of INT407 cells and HT29 cells decreased after treatment with high concentration of dietary digestive products, especially in HMb group. Moreover, the cell viability of INT407 cells cultured for 24 h was significantly lower than that of 12 h (P < 0.05), while HT29 cells had no significant difference between 12 h and 24 h (P > 0.05). At 0.25 and 1 mg/mL, the treatment of cells with myoglobin digestion products increased cell apoptosis, and the effect on HT29 cells was greater than that on INT407 cells. The higher the concentration of digestion products, the higher the cell apoptosis, especially early apoptosis. When INT407 cells were cultured with 0.25 and 1 mg/mL myoglobin digestion products, G2 phase in the cell cycle was prolonged, G1 and S phases were shortened. The results of HT29 cells cultured with 1 mg/mL myoglobin digestion products were consistent with them. When HT29 cells were cultured at 0.25 mg/mL, the S phase in the cell cycle was prolonged and the G1 phase was shortened. In addition, with the increase of myoglobin content, the production and accumulation of MDA in cells, SOD activity was significantly inhibited. The above results indicated that high myoglobin resulted in arrested cell cycle, induced lipid peroxidation, and inhibited the activity of antioxidant enzymes in INT407 cells and HT29 cells. In conclusion, the damage to intestinal mucosal barrier caused by high-dose myoglobin is caused by excessive oxidative stress induced by heme, while low-dose myoglobin not cause changes in intestinal homeostasis.