College of Life Science, Northeast Agricultural University, Harbin 150030, China
Persistent inflammation, immunosuppression, and catabolic syndrome (PICS) are the main causes of poor outcomes in patients transferred out of ICU. Hyaluronic acid has a potential function to alleviate PICS, and, as a widely accepted food additive, HA can be consumed through food products such as dairy products, greatly increasing patient medical compliance. This study evaluated the remission effect of PICS model mice after oral administration of HA with different molecular weights. The results showed a nonlinear relationship between HA molecular weight and its effect of intervention with symptoms in PICS mice and the high molecular weight HA (HMW-HA) was more effective in relieving symptoms in PICS model mice. Therefore, we believe that HMW-HA can be used to prepare functional foods for the purpose of intervening with PICS disease.
Hyaluronic acid (HA) is a new functional food additive which has the potential to ameliorate persistent inflammation, immunosuppression and catabolism syndrome (PICS), but the biological effects of HA with various molecular weights differ dramatically. To systematically investigate the efficacy of HA in altering PICS symptoms, medium-molecular-weight (MMW) HA was specifically selected to test its intervention effect on a PICS mouse model induced by CLP through oral administration, with high-molecular-weight (HMW) and low-molecular-weight (LMW) HA also participating in the experimental validation process. The results of pathological observations and gut flora showed that MMW HA rapidly alleviated lung lesions and intestinal structural changes in PICS mice in the short term. However, although long-term MMW HA administration significantly reduced the proportions of harmful bacteria in gut flora, inflammatory responses in the intestines and lungs of PICS mice were significantly higher in the MMW HA group than in the HMW HA and LMW HA groups. The use of HMW HA not only rapidly reduced the mortality rate of PICS mice but also improved their grip strength and the recovery of spleen and thymus indices. Furthermore, it consistently promoted the recovery of lung and intestinal tissues in PICS mice, and it also assisted in the sustained restoration of their gut microbiota. These effects were superior to those of LMW HA and MMW HA. The experimental results indicate that HMW weight HA has the greatest potential to be an adjunct in alleviating PICS as a food additive, while the safety of other HAs requires further attention.
Persistent inflammation, immunosuppression, and catabolism syndrome (PICS) is a multifaceted disease caused by immune disorders, and no effective treatment is available because of its complex pathogenesis. The main clinical intervention is to regulate diet and nutrient intake. Sepsis is a life-threatening medical condition caused by infections, burns, and other factors with a mortality rate of 25–30%. Although the mortality rate of sepsis has dramatically decreased with advances in medical treatments, sepsis survivors have serious health problems, and the prognosis is poorer for those who experience PICS.
Hyaluronic acid (HA), which is also known as hyaluronate and hyaluronan, is a non-sulfated glycosaminoglycan and endogenous mucopolysaccharide. It is composed of repeating disaccharide units of β-1,4-D-glucuronic acid and β-1,3-N-acetylglucosamine. As an important component of the extracellular matrix, several preclinical and clinical experiments have shown good efficacy regarding the anti-inflammatory, wound healing, and antiangiogenic properties as well as the safety profile of hyaluronic acid. In addition to clinical application, HA has been used in the food processing industry as an egg white substitute as early as 1942. To date, lots of countries including China, the European Union and the USA have accepted the usage of HA as a food ingredient or dietary supplement. However, limited research studies are available that have explored the functional benefits of HA in dairy and food applications. HA has been proved that this molecule has strong immunomodulatory activities and regulates the intestinal system; thus, there is a huge potential for the oral delivery of HA through foods to gain health benefits. Moreover, as a substrate for gut microbiota, HA may have beneficial impacts on the host by modulating the composition of the gut microbiota. High-molecular-weight (HMW) HA prevents its free transit through the intestinal wall, which suggests that the HMW HA may had a greater impact on the gut microbiota composition.
Interestingly, the functional attributes of products containing HA would still depend upon the MW the HA used. Our previous study demonstrated significantly different effects of high-molecular-weight (HMW) HA and low-molecular-weight (LMW) HA on PICS. HMW HA was better than LMW HA in relieving inflammation, whereas LMW HA was superior to HMW HA in improving the gut microbiota composition after long-term use. However, the lack of experimentation with medium-molecular-weight HA directly restricts the practical comprehensive application of HA. Thus, it aroused our interest to explore whether the medium-molecular-weight (MMW) HA has therapeutic advantages over the above HAs.
In the present study, we established a PICS mouse model in accordance with the method described by Amanda et al. Typical HMW HA (1600 kDA), MMW HA (100 kDA), and LMW HA (3 kDA) were administered to this mouse model. By observing changes in the physiological indicators, pathology, and gut flora, we compared the effects of HMW, MMW, and LMW HA on PICS mice and investigated the feasibility of HA as a functional food additive in the dietary regulation of PICS patients.
To evaluate the therapeutic efficacy of HA of different molecular weights in the mouse models of PICS, we examined changes in the survival, grip strength, and organ coefficients in each experimental group. As Figure 1 shows, the risk of death in PICS mice gradually decreased with the recovery time but was still present in the early stage. After HA treatment, the mortality of treated mice was reduced by various degrees in all groups. The organ coefficient results on day 10 showed that with the increase in HA molecular weight, the spleen index was closer to the WT group. However, the grip strength test on day 30 showed that the molecular weight of HA was positively correlated to the recovery of grip strength in mice. Further experiments are needed to determine the reason for this result and to assess the health status of mice.
Figure 1. Effects of 3 kDA-HA (PICS mice model treated with 3 kDA-HA), 100 kDA-HA (PICS mice model treated with 100 kDA-HA), and 1600 kDA-HA (PICS mice model treated with 1600 kDA-HA) administration intervention on (A) survival rates; (B) grip strength; (C) spleen index; and (D) thymus index of PICS mice. Data are presented as mean ± standard deviation. * p< 0.05, ** p< 0.01 and *** p< 0.001 compared to WT group (mice underwent a sham surgery and treated with saline). #p< 0.05, ##p< 0.01 compared to CK group (PICS mice model treated with saline).
We examined the lung lesions in the mice by using pathological sections. The results showed that short- and long-term pathological changes in the lungs of PICS mice were observed. The CK group had progressively deeper lung lesions with a longer disease duration, together with a disorganized lung architecture and remarkably thickened alveolar walls, and some alveoli had disappeared. Moreover, a large number of inflammatory cells infiltrating into alveolar and interstitial cavities were visible, which was accompanied by notable pathological features such as alveolar hemorrhaging and alveolar wall rupture. Mice in the 3 kDA-HA group showed a small amount of alveolar hemorrhaging and slight thickening of the alveolar wall in the short term. However, they still had more inflammatory cell infiltration in the long term. Conversely, mice in the 100 kDA-HA group did not have severe lesions in the short term. However, lung lesions worsened after prolonged administration of 100 kDA-HA along with a massive infiltration of inflammatory cells and tissue hemorrhaging. Mice in the 1600 kDA-HA group had more normal findings with no large or obvious lesions observed.
Figure 2. Results of H&E staining of PICS mice lungs on days 10 and 30 in each group after intervention with 3 kDA-HA, 100 kDA-HA, and 1600 kDA-HA administration. All representative histological images were taken at a magnification of ×200.
Pathological changes in the gut were also the focus of examination in this study. After 10 days of HA administration, three mice were randomly selected from each group, and their intestines were harvested and observed for morphology. The intestinal system of mice in the CK group had pronounced atrophy and was more prone to rupture. Additionally, multiple swellings were observed in the intestinal wall, and the intestines tended to be black, which was probably because of long-term under-perfusion of the intestinal system or intestinal obstruction. Although HA groups also had swellings in the intestinal wall, the gut was essentially smooth and intact, and its color was similar to that in the WT group.
Figure 3. Results of macroscopic intestinal morphology at day 10 and H&E staining of intestinal tissues from PICS mice in each group at day 10 and 30 after intervention with 3 kDA-HA, 100 kDA-HA, and 1600 kDA-HA administration. All representative histological images were taken at a magnification of ×200.
On days 10 and 30, the proximal and distal ends of the colon were harvested for H&E staining. In the short term, the CK group showed an atrophy of the lamina propria with a significant decrease in thickness, a decrease in the number of goblet cells and absorptive cells in crypts, and pronounced destruction of the upper structure of crypts. Conversely, the 3 kDA-HA group lacked the semilunar fold. After 30 days of HA administration, the intestinal structure had remarkably recovered in the 3 kDA-HA group, but it continued to deteriorate in the CK group. The 1600 kDA-HA group showed good recovery, which was superior to that in the other groups after day 10, and the intestinal structure subsequently became similar to that of the WT group. Changes in the intestinal structure were most notable in the 100 kDA-HA group. Recovery of the intestinal structure was far superior in the 100 kDA-HA group compared with the other groups on day 10. However, the intestinal structure in the 100 kDA-HA group showed significant inflammatory cell infiltration on day 30 along with disruption of the tissue structure.
To clarify the correlation of the effect of HA molecular intervention in PICS and changes in gut microbiota, sequencing results were analyzed by clustering and taxonomy. The number of flora in the 1600 kDA-HA group gradually returned to normal, but the number of operational taxonomic units in the 3 kDA-HA and 100 kDA-HA groups was significantly lower than that in normal mice and lower than those in the other two groups of PICS mice. Additionally, alpha-diversities in the 3 kDA-HA and 100 kDA-HA groups were closer to that in the WT group compared with the 1600 kDA-HA and CK groups.
Figure 4. Effects of 3 kDA-HA, 100 kDA-HA and 1600 kDA-HA on the abundance and diversity of mice intestinal microbiota at day 30. (A) Venn diagram of microbial abundance; (B) microbial alpha diversity; (C) microbial beta diversity.
Beta-diversity analysis was performed on the flora composition and phylogenetic information of multiple samples. During the initial period, the intestinal flora of all four PICS groups differed significantly from that of normal mice. However, the flora composition of the 3 kDA-HA, 1600 kDA-HA, and CK groups tended to recover toward the flora composition of the WT group. Meanwhile, the flora composition differed significantly between the 100 kDA-HA and WT groups.
Changes in the proportion and abundance of gut flora were most pronounced in the 100 kDA-HA group. The proportions of harmful bacteria were significantly reduced, including Escherichia Shigella in class Gammaproteobacteria, Streptococcaceae in class Bacilli, Blautia in class Clostridia, and Erysipelotrichaceae in class Erysipelotrichia. However, the proportions of beneficial bacteria, especially those involved in regulating the immune response (e.g., Ruminococcaceae and Clostridiaceae_1 in class Clostridia) were significantly increased. The proportions of gut microbiota in the other groups were not significantly different from those in a previous study.
Figure 5. Effects of 3 kDA-HA,100 kDA-HA and 1600 kDA-HA on inter-group differences in mice intestinal microbiota and the abundance of dominant bacteria at different taxonomic levels were investigated at day 30. Community structure at di
