The results of this study revealed that MCHs markedly decreased somatic cell count after 7 days of treatment. Although we did not observe any changes in bacterial community richness by the Chao indices, the Simpson index showed a significant decrease in the bacterial diversity. In particular, on day 7, the relative abundance of Firmicutes was significantly decreased, and that of Proteobacteria was significantly increased compared to levels on day 1. It has been reported that the richness and diversity of the milk microbiome reflects the health of the cows and functional performance of the organs [16–18]. Antibacterials are commonly used to treat bovine mastitis, but their efficacy is unclear . That is why we chose to test the plant-derived compound, matrine, as an intramammary treatment loaded into chitosan hydrogels to see if it would alter the milk microbiota and improve mammary gland function.
Measurement of the number of somatic cells in milk is an internationally recognized test for the detection of mastitis. A SCC of more than 200,000 per ml is considered to be evidence of disease. Recent investigations have shown that the milk microbiota of dairy cows are usually closely associated with the SCC [21, 22]. This suggests that modulation of the udder ecosystem through the microbiota could help to maintain homeostasis and enhance the mammary gland’s defense mechanism [23, 24]Consistent with these reports, we observed that the udder SCC was significantly increased by in mastitis by common pathogens like Staphylococcus, but was markedly decreased after perfusion of the udder with matrine-chitosan hydrogel. This is in line with previous studies showing that chitosan hydrogels decreased the SCC in milk from dairy cows. The therapeutic synergy achieved by loading the chitosan hydrogel with matrine suggests potential application in mastitis protection and treatment. The mechanism by which the milk microbiota influences inflammatory and immune responses to decrease the SCC still needs further investigation.
The next generation of high-throughput DNA sequencing methods together with updated bioinformatics software is now being used for the in-depth assessment of microbial communities to elucidate how bacterial activity and metabolites affect human and animal health . We found that the microbial diversity in milk was dramatically decreased after one week of intramammary infusions of matrine-chitosan hydrogels. The relative abundance of Proteobacteria was significantly increased, while Staphylococcus numbers decreased significantly. Staphylococci are internationally recognized as one of the most prevalent mastitis pathogens, accounting for about 70% of the cases .. Moreover, it has been reported that Proteobacteria are significantly more abundant in healthy cows than those with mastitis. Previous studies also showed that the numbers of Proteobacteria after non-antibiotic hydrogel treatment increased significantly , indicating that hydrogel therapy can effectively return the milk microbial environment to normal diversity and defend against disease. Our investigation provides mechanistic insights into how the microbiota respond to MCH may help in the development of novel prophylactic and therapeutic products as alternatives to antibiotics in dairy cows. Our data are also important for understanding how the regulation of biosynthesis by milk microbiota influences udder health and defense against mastitis. The milk microbiota are usually closely associated with the SCC [21, 22]. suggesting that the udder microbiota plays an important part in modulation of the ecosystem and maintenance of homeostasis to resist mammary gland infections [23, 24]
Metabolomics is a comparatively new research method that has been widely used in the detection of mastitis in recent years due to its more comprehensive test results. Previous studies suggested that metabolomics could provide a more complete understanding of an animal’s physiology and biochemistry .. The metabolomics data from this study highlight the potential function of matrine-chitosan hydrogels in modulating the metabolite levels, which were found to be enriched in sphingolipid metabolic pathways, phenylalanine, glycerophospholipid, and starch and sucrose metabolism. A previous report suggested that sphingolipid metabolites acted as signaling molecules to regulate a diverse group of cellular processes, particularly those related to immunity, inflammation and inflammatory disorders .. We speculate that this activity might be associated with the decrease in SCC caused by matrine-chitosan hydrogel injection. The metabolic differences we observed gave us a further insight into how MCH affects metabolite levels before and after mastitis treatment, and showed that alterations in the milk metabolome could be used to reveal the therapeutic mechanism of matrine for mastitis treatment and in the recovery of milk production in dairy cows.
We found significant changes in concentration of a number of sphingolipids, glycerolipids, fatty acyls, glycerophospholipids, and organo-oxygen compounds that significantly differed from day 1 to day 7. Sphingolipids are major components of cell membranes and are considered to be widely involved in important processes such as cell aging and apoptosis .. Glycerolipids  and fatty acyls  are both important components of cell membranes. Previous research found that bacterial invasion can induce inflammatory reactions and cause oxidative damage during the development of the breast , accompanied by apoptosis .[34, 35]. The reduction in sphingolipids, glycerolipids, and fatty acyls seen in this study indicates that apoptosis caused by inflammatory reactions may have been weakened. The decrease of organooxygen compounds suggests that the oxidative damage caused by inflammatory reactions may also have been reduced. The metabolites were enriched in sphingolipid and glycerophospholipid metabolism, which are both related to apoptosis pathways.[37, 38]. Therefore, we conclude that MCHs can inhibit apoptosis by blocking oxidative damage, thereby reducing inflammatory changes to the udder.
It is worth noting that L-phenylalanine is involved in many pathways that are significantly changed in this study (table 3). L-phenylalanine is an essential amino acid and a precursor for the synthesis of antiviral and anticancer drugs as well as new sweeteners. It can also be a marker of inflammatory reactions and affect the body's immune response . Here, the content of L-phenylalanine was significantly increased on day 7, which means that the immune defenses in the mammary gland may have been effectively improved. Previous study  found that tryptophan and phenylalanine are used in the body to synthesize serotonin, L-DOPA derivatives and 5, 6, 7, 8-tetrahydrobiopterin (BH4), which compounds can affect the severity of inflammation. In the study of Smith ,, tyrosine and phenylalanine participated in the regulation of TLR4 signaling pathways, thereby influencing the degree of inflammation. Phenylalanine also is part of the glycolytic and liposynthetic pathways.[40, 41]. It has been reported that phenylalanine deficiency can severely affect protein metabolism in the breast and compromise the health of the organ.. Phenylalanine is an essential amino acid in milk and is also the precursor of tyrosine, which is one of the main amino acids in milk.. We know that untreated bovine mastitis results in reduced milk fat and milk protein content; therefore, the increase in L-phenylalanine by MCH injection could be effective in restoring normal milk production during lactation.
The milk metabolites produced by MCH injection were found to have an intimate relationship with sphingolipid and glycerophospholipid metabolism. A previous study suggested that the sphingolipid metabolic pathways participate in a variety of immune-related signal transductions, inflammation and inflammatory disorders . The sphingolipid metabolism-related pathways have many functions, such as regulating cell adhesion and cellular immunity, activating cancer repressors, regulating apoptosis, and modulating immune function and the inflammatory response  We speculate that this activity might be associated with the decrease in SCC caused by MCH injection. Furthermore, Cowart et al.  showed that an imbalance in the pathway for sphingolipid metabolism can cause ketosis, mastitis, and metritis in cows. It has been proved that sphingolipid metabolites, especially ceramide and sphingosine-1-phosphate, can regulate a variety of biochemical processes important in immune, inflammation and inflammatory diseases. In the present study, we found that MCHs could significantly regulate the sphingolipid metabolism-related pathways to improve inflammation; thus, it is reasonable to believe that MCHs could effectively treat bovine mastitis.
Despite the documented health-promoting properties of matrine and chitosan, scientific evidence for the efficacy of MCHs in dairy cows is limited. Our study affords many useful insights, but the effect of MCHs on immune regulation still requires further study. Another limitation relates to the mechanistic links involved in the observed significant changes in the microbiota and biomarker metabolites as a result of MCH injection that will need further exploration to be clearly understood. Previous studies suggested that the antimicrobial activity of chitosan can stimulate the innate immune response and hasten the involution process of the mammary gland. It is not known whether matrine-chitosan complexes could be used in the circulation to regulate the immune system and inhibit inflammation, but our data indicate that future research in this area is strongly warranted.