Tissue Distribution Of Polymannuronic Acid In Mice And Its Effects On Gut Microbiota

Polymannuronic acid(PM)is an alginate oligosaccharide with a definite structure obtained from brown algae.PM has various biological activities and significant potential for development.Current studies primarily focus on PM degradation mode,derivation,and pharmacological activity.However,less attention has been paid to PM absorption,tissue distribution,pharmacokinetics,and intestinal floral effect in vivo.These challenging aspects should be solved in PM development and industrialization.The present study used fluorescein isothiocyanate(FITC)for PM labeling to improve detection sensitivity and stability based on the in vitro mechanism of PM transport across small intestinal mice epithelial cells.PM distribution in tissues and pharmacokinetics after different administration modes of FITC-PM were studied.Finally,16S rRNA gene high-throughput sequencing was performed to analyze the long-term PM intragastric administration effects on the microflora inside the jejunum,ileum,colon,and feces of mice,followed by experimental validation.This study provided the following innovative results:1.Stable FITC-PM was obtained with improved PM detection sensitivity.High-purity PM was prepared using acid degradation,multiple pH classification,ion exchange chromatography,and gel chromatography with sodium alginate as the raw material.The PM had an average molecular weight of 16.83 kDa and an average polymerization degree of 95.FITC-PM was prepared by the reaction of tyramine with the hemiacetal hydroxyl group of PM,which added the secondary amine group,and the nucleophilic reaction,which linked the FITC.The obtained FITC-PM was verified using agarose gel electrophoresis.Moreover,the FITC-PM synthesized using FITC labeling had high stability in a wide range of pH(pH 2-12),artificial gastric fluid and artificial intestinal fluid,and can be used to study its distribution in tissues and pharmacokinetics in vivo.2.Thein vivo PM distribution and pharmacokinetics were.clarified using quantitative analysis of FITC-PM after tail.intravenous injection or intragastric administration,demonstrating the liver and kidney targeting of PM.Thus,the intestinal tract could absorb part of FITC-PM into the bloodstream after intragastric administration.The in vivo distribution of FITC-PM after tail intravenous injection(50 mg/kg)revealed that FITC-PMfirst entered and distributed mainly in the kidney and liver,with a maximum value of 494.50± 73.52and 236.67±50.94 μg/g respectively at 0.5 h.Additionally,intravenous injected FITC-PM was distributed at relatively low concentrations in the heart,spleen,and lungs.Based on the pharmacokinetic study,FITC-PM was rapidly eliminated in the blood after tail vein injection into the mouse.PM concentration achieved the maximum value of 235.17 ± 26.06 μg/mL at 0.5 h.Then,the concentration indicated a downward trend,and after 12 h,FITC-PM was hardly present in the blood.FITC-PM had different elimination rates among different mice,with significant individual distinction.For FITC-PM,the elimination rate constant(Kel)was 0.24 ± 0.05,the half-life in mice was 2.85 ± 0.41 h,and the maximum blood concentration was 235.17±26.06 μg/mL.Moroever,the area under the concentration-time curve(AUC0-∞)was 631.48±180.63 μg/mL·h,the area under the first moment curve(AUMC0-∞)was 1843.15±301.19 μg/mL·h2,the average retention time was 2.92 ± 0.42 h,and the clearance rate was 79.18 ± 19.36 mL/h.After intragastric administration(100 mg/kg),tissue distribution revealed that some FITC-PM could be absorbed through the intestine into the bloodstream.The distribution of FITC-PM absorbed through intestinal tract was basically the same as that of intravenous administration,mainly in liver and kidney,with the highest concentration of 95.35±28.20 μg/g in liver at 2 h and 192.96±21.68 μg/g in kidney at 4 h.Based on the pharmacokinetic studies,the Kel of FITC-PM was 0.25±0.06,the half-life in mice was 2.73 ± 0.53 h,and the maximum blood concentration was 95.62± 23.37 μg/mL.Additionally,theAUC0-∞ was 508.33±143.82 μg/mL-h,the AUMC0-∞was 2645.83 ± 520.47 μg/mL·h2,the average retention time was 5.20±0.53 h,and the clearance rate was 196.72±35.29 mL/h.Experimental results indicated that the kidneys and livers of mice had a strong PM uptake capacity,and the kidneys excreted the PM irrespective of tail intravenous injection or intragastric administration.PM showed a specific accumulative effect in the kidneys.3.16S rRNA gene high-throughput sequencing analysis indicated significant differences in microorganism abundance and diversity in different intestinal segments and feces of mice after long-term PM intragastric administration.Thus,the PM could significantly decrease the abundance of conditioned pathogen,such as Escherichia,in the intestinal tract.Mice were intragastrically administered PM for 30 days in high-dose(400 mg/kg)and low-dose(100 mg/kg)groups.This was followed by the aseptic collection of the contents in the jejunum,ileum,colon,and intestinal segment of mice and feces at different times.16S rRNA gene was extracted for high-throughput sequencing(n=6)of the V3-V4 regions.The sequencing results were as follows:(1)OTU statistics and Alpha diversity analysis revealed that 30 days of intragastric administration with different PM doses could enhance the flora abundance in the jejunum,ileum,and colon.However,they showed no significant effect on the flora diversity of each intestinal segment.PM affected the fecal flora of mice in a time-dependent manner.The fecal flora diversity changed with PM intragastric administration time extension,becoming the most significant on Day 30.The effects of different PM doses on the intestinal and fecal microflora of mice,depending on Beta diversity analysis,were the same after 30 days of intragastric administration.The low-dose group mainly induced microflora along the plsda2 direction.In contrast,the high-dose group induced microflora along the plsdal and plsda2 directions.(2)High-and low-dose PM intragastric administration for 30 days could promote the microflora balance in different intestinal mice segments based on the B/F index.Regarding intragastric administration time,PM first caused fecal flora imbalance with intragastric administration time.However,the fecal flora in the highand low-dose groups recovered balance on Day 30,becoming superior to the control group.(3)Long-term PM intragastric administration significantly reduced the conditioned pathogen Escherichia abundance in the jejunum,ileum,colon,and feces.The dominant species correlation and the KEGG functional difference analyses of flora indicated that the inhibitory effect of PM on Escherichia was gradually enhanced with intragastric administration time.PM did not inhibit Escherichiadirectly,but by regulating the abundance of other microorganisms,which collectively led to the decrease of Escherichia abundance.(4)After 30 days of PM intragastric administration,Prevotella became the dominant bacterial community within the colon and feces of mice,showing Prevotella intestinal type.Moreover,Prevotrlla abundance was significantly elevated in both high-and low-dose groups.Thus,the PM could induce the Prevotrlla intestinal type production.(5)KEGG functional difference analysis of the different intestinal segments and fecal flora on Day 30 revealed that long-term PM intragastric administration could promote lipid metabolism in intestinal and fecal flora except for the colon.This was verified by determining the serum total TC,FC,and cholesterol ester contents of mice in each group.Thus,PM can reduce serum TC,cholesterol ester,and liver index according to a concentration-dependent manner.(6)The analysis of bacterial dominant species composition and KEGG functional difference revealed that high-dose PM intragastric administration could cause colon-intestinal inflammation by enhancing the abundance of various floras in the jejunum,ileum,colon,and feces.The experimental verification results indicated that high-dose PM could elevate the content of the signature inflammatory factors,viz.,LBP and IL-1β.The dose had an essential effect on the efficacy of PM,with low-dose PM having a superior effect on intestinal flora than high-dose PM in general.