| Hyaluronan(HA)is a naturally occurring glycosaminoglycan(GAGs)composed of D-glucuronic acid and N-acetylglucosamine.It is a crucial structural component of the extracellular matrix and plays a vital and abundant biological role in vivo.The biological function of HA is closely associated with its molecular weight,yet there remains a lack of understanding regarding the absorption,distribution and functionality of HA with varying molecular weights after oral administration.The orally administered HA that enters the bloodstream is selectively taken up by tissue cells to perform its designated function,however,there remains a dearth of research and data supporting the ability of HA with varying molecular weights to penetrate cell membranes.Therefore,considering the aforementioned scientific issues,this study conducted a three-tiered research approach:firstly,investigating the absorption and tissue distribution of HA with varying molecular weights following oral administration.Subsequently,examining the membrane permeability of HA with different molecular weights through fluorescence-labeled HA combined with super-resolution microscopic imaging technology.Finally,the effects of HA on skin and liver with vary ing molecular weights were investigated using a UV cell injury model and an acute liver inj ury model in mice.The primary research topics are delineated as follows:1.Research on the relationship between molecular weight and oral absorption ability of HARats were first given a single gavage of 30 kDa,600 kDa,and 1,250 kDa HA to mimic a single oral absorption of HA.The results showed that after oral administration of HA,the content of HA in serum of rats increased first,then decreased,and then increased again,which may correspond to the process of absorption,degradation and resynthesis of HA after oral administration.Secondly,we also found that the rate of oral absorption of HA slowed down and the serum HA content increased with the increasing molecular weight of HA.Subsequently,the rats were treated with 30 kDa,600 kDa,and 1,250 kDa HA by gavage for 15 consecutive days,and then were observed for another 15 days after stopping gavage to simulate long-term oral absorption of HA.The results showed that after continuous oral administration of HA,the serum HA content increased rapidly at first,then decreased slightly and remained stable at a level higher than that of the blank control group.After stopping oral administration of HA,the content of HA in serum quickly returned to normal values,indicating that long-term oral administration of HA can increase the content of HA in serum and supplement the deficiency of endogenous HA.Subsequently,the mice were treated with 30 kDa,600 kDa,and 1,250 kDa HA by gavage for 28 consecutive days,and the effects of oral administration of HA with different molecular weights on HA content in tissues and gut microbiota composition were further analyzed.The results showed that long-term oral administration of HA increased the content of HA in skin and liver and HAase in liver of mice.The mice orally administered with 600 kDa HA had the highest HA content in skin and liver and the highest HAase content in liver(P<0.01),while the mice orally administered with 1,250 kDa HA had the lowest HA content in skin and liver.At the same time,oral administration of HA also increased the abundance of intestinal flora and decreased the content of Helicobacter pylori in feces of mice(P<0.05).These results suggested that long-term oral administration of HA could increase the content of HA in tissues and induce more HAase expression in the liver.Of the three molecular weights selected,600 kDa HA had the strongest ability to distribute into tissues.The above studies showed that oral administration of HA could increase the content of HA in serum,and the molecular weight of HA affected the absorption effect of oral administration,which further affected the change of HA content in serum and the distribution of HA in organs.2.The relationship between the molecular weight of HA and its transmembranous absorption capacityHA of 30 kDa,200 kDa and 1,250 kDa was labeled with 6-aminofluorescein(FA),and the products were characterized and the fluorescence labeling efficiency was analyzed.HepG2 and HaCaT cells were used to analyze the difference in the transmembrane absorption of HA with different molecular weights and the intracellular distribution of exogenous HA.Superresolution microscopy and fluorescence microscopy showed that 30 kDa,200 kDa and 1,250 kDa HA could be incorporated into HcpG2 and HaCaT cells after fluorescently labeled,but there was no significant correlation between the intracellular fluorescence intensity and molecular weight,indicating that there was no significant difference in the uptake ability of the above three molecular weights of HA.Meanwhile,the molecular weight of HA is higher,the sugar chain is longer,and the number of fluorescent molecules is higher.Therefore,from the perspective of the amount of HA ingested by cells,the smaller the molecular weight of HA sugar chain is,the more efficient it is to transport through the cell membrane into the cells.The above results demonstrate that the cells have a certain uptake capacity for HA,and HA absorbed into the blood after oral administration can enter the tissues through cell-specific uptake.3.The correlation between the molecular weight of HA and its protective effects on skin and liver.Firstly,a UV-damaged HaCaT cell model was constructed to study the effects of HA with different molecular weights on UV-damaged cells.The results showed that HA with different molecular weights significantly increased the survival rate of UVdamaged HaCaT cells(P<0.01).The effect of HA with different molecular weight was 2000 kDa≈1,250 kDa>200 kDa≈30 kDa>600 kDa.At the same time,super-resolution microscopy results showed that HA-FA colocated with mitochondria in the cells,and HA-FA incubation significantly improved the state of mitochondria in the UV-damaged cells,indicating that HA could be transported to the vicinity of mitochondria to repair UV-damaged mitochondria and improve cell survival rate.Subsequently,the protective effect of long-term oral administration of HA on the liver was studied through a mouse model of alcoholic acute liver injury.The results showed that the contents of TG and MDA in the liver of mice treated with 30 kDa and 1.250 kDa HA were lower than those of the model group,and the GSH content was increased,and the effect of 30 kDa HA on MDA was more significant.The effect of 1,250 kDa HA on GSH was more significant(P<0.05).These results suggest that oral administration of HA of 30 kDa and 1,250 kDa can ameliorate acute liver injury in mice,and different molecular weights have different effects on TG,MDA and GSH.HA of 600 kDa has no obvious effect on acute liver injury,and its mechanism needs further study.In conclusion,oral administration of HA could increase HA content in serum and tissues.HA with low molecular weight was absorbed quickly,HA with high molecular weight was absorbed in blood,and HA with middle molecular weight was distributed in tissues.HA of different molecular weight can enter the cell through the cell membrane.The molecular weight of HA is not related to the uptake ability,but it affects the uptake efficiency.The smaller the molecular weight of HA,the higher the uptake efficiency.This provides a basis for the selection of molecular weights for the development of oral HA products.At the same time,in the study of UV-damaged cells and acute liver injury models,it was found that HA played different roles in UV damage repair and liver injury protection due to different molecular weights,but the effect was not proportional to the absorption capacity,indicating that the effect of HA was not only related to the content of HA distributed in tissues and cells,but also there may be other mechanisms.Further research is needed. |
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