Introduction
Chronic pancreatitis (CP) is a debilitating inflammatory condition of the pancreas characterized by progressive and irreversible damage. It leads to exocrine and endocrine insufficiencies, chronic pain, and severe complications such as diabetes mellitus, cholangitis, ascites, and even pancreatic carcinoma. The etiology of CP is multifactorial, with lifestyle factors such as alcohol consumption, genetic predispositions, and environmental influences playing significant roles. Oxidative stress has been implicated as a critical mechanism in the pathogenesis of CP, causing pancreatic inflammation and fibrosis.
Recently, the gut microbiota's role in various diseases, including CP, has gained considerable attention. Dysbiosis, or the imbalance in gut microbiota, is associated with inflammatory conditions and metabolic disorders. Polysaccharides have emerged as potential therapeutic agents due to their prebiotic effects and ability to modulate gut microbiota. Inonotus obliquus, commonly known as Chaga mushroom, is a medicinal fungus with extensive biological properties, including antioxidant and anti-inflammatory activities. This review examines a
clinical study (PMC5309192) investigating the effects of Inonotus obliquus polysaccharide (IOP) on chronic pancreatitis in a mouse model, focusing on its ability to reduce inflammation and tissue damage.
Materials and Methods
Chemicals and Reagents
The study utilized Inonotus obliquus (CFCC83280) provided by Harbin Baykaltai Bioengineering Co. LTD of China. Various chemicals and reagents, including anion-exchange DEAE cellulose columns, Sephadex G-200 gel, chloroform, butanol, ethanol, DDC, and commercial assay kits for GSH-PX, TAOC, TNF-α, TGF-β, lipase, and trypsin, were employed in the experiments. DNA mini stool kits and MetaVx™ library preparation kits were used for genetic and sequencing analyses.
Preparation of IOP
IOP was prepared following a previously established procedure. The dried sclerotia of Inonotus obliquus were ground into powder and extracted with distilled water at 60°C for 2.5 hours. The supernatant was concentrated and treated with Sevag reagent to remove proteins. The mixture was then precipitated with ethanol, and the crude IOP was purified using an anion-exchange DEAE cellulose column and a Sephadex G-200 gel column. The resulting IOP was a homogeneous polysaccharide with a molecular weight of 32.5 kDa and a polysaccharide content of 98.6%.
Toxicity Test
A toxicity test was conducted to ensure the safety of IOP. Male ICR mice were administered IOP at a dose of 1 g/kg body weight by oral gavage thrice a day. The control group received a saline solution. The mortality and side effects of the mice were monitored for 72 hours, and no deaths or adverse effects were observed.
Experimental Design
The study involved six groups of mice, each consisting of ten individuals: three IOP-treated groups (IOP-L, IOP-M, and IOP-H), a Qingyilidan granule-treated group (PC), a model control group (MC), and a standard control group (NC). Except for the NC group, all mice received intraperitoneal injections of DDC (10%, 0.5 g/kg body weight) twice a week for four weeks to induce CP. The IOP-treated groups were administered IOP at dosages of 0.1 g/kg/day (IOP-L), 0.2 g/kg/day (IOP-M), and 0.4 g/kg/day (IOP-H) for four weeks starting from the second week of DDC injection. The PC group received Qingyilidan granules at a 3.7 g/kg/day dose. After the five-week experiment, all mice were sacrificed via cervical dislocation.
Measurement of Biochemical Parameters
Biochemical parameters were measured using commercial assay kits, including GSH-Px, TAOC, TNF-α, TGF-β, lipase, and trypsin. Pancreatic tissue homogenates and blood samples were analyzed to determine the levels of these parameters.
Genomic DNA Preparation and Sequencing
Fecal samples were collected from each group at the end of the experiment. Total DNA was extracted using a method described by Yu and Morrison. DNA libraries were prepared using the MetaVx™ library preparation kit, and sequencing was performed on an Illumina MiSeq instrument.
Bioinformatics and Statistical Analysis
The sequences were clustered into operational taxonomic units (OTUs) using a 97% identity cut-off. Diversity and richness analyses were performed using OTUs. Partial least square discriminate analysis (PLS-DA) identified key OTUs based on their contribution to the biochemical characteristics. Pearson correlation analysis evaluated the correlations between biochemical characteristics and gut microbiota at the phylum level.
Results
Toxicity Test
The toxicity test revealed no deaths or adverse effects in mice treated with IOP, indicating its safety for further experiments.
Biochemical Characteristics
GSH-Px and TAOC Levels
The levels of pancreatic GSH-Px and TAOC were significantly reduced in the MC group, indicating oxidative damage induced by DDC. However, IOP treatment increased GSH-Px and TAOC levels in a dose-dependent manner. The highest levels were observed in the IOP-H group, comparable to the NC group, suggesting that IOP effectively prevented oxidative damage in the pancreas.
TNF-α and TGF-β Levels
Serum TNF-α and TGF-β levels were markedly elevated in the MC group, reflecting prolonged inflammatory responses and pancreatic injury. IOP treatment significantly reduced TNF-α and TGF-β levels compared to the MC group. The reductions were more pronounced in the IOP-H group, indicating that IOP attenuated inflammation in CP mice.
Lipase and Trypsin Levels
Serum lipase and pancreatic trypsin levels were highest in the MC group, indicative of severe pancreatic damage. IOP treatment significantly lowered lipase and trypsin levels, with the IOP-H group showing the most substantial reductions. This suggests that IOP mitigated pancreatic tissue damage in CP mice.
Gut Microbiota Composition and Diversity
High-throughput sequencing revealed significant alterations in CP mice's gut microbiota composition and diversity. The MC group exhibited reduced microbial diversity, as indicated by lower Shannon diversity index and Chao1 estimator values. IOP treatment modulated the gut microbiota, increasing the proportion of Bacteroidetes and decreasing Firmicutes at the phylum level. Bacteroidetes positively correlated with GSH-Px and TAOC levels, while Firmicutes correlated with TNF-α, TGF-β, and lipase levels. These findings suggest that IOP restored gut microbiota homeostasis in CP mice.
Discussion
Antioxidative Effects of IOP
The study demonstrated that IOP significantly increased GSH-Px and TAOC levels in CP mice, indicating its potent antioxidative properties. GSH-Px is a crucial enzyme in superoxide degradation, and its reduced levels are associated with oxidative stress-related diseases, including CP. TAOC reflects the nonenzymatic antioxidant defense capacity. The dose-dependent increase in GSH-Px and TAOC levels with IOP treatment suggests that IOP effectively mitigates oxidative stress in CP.
Anti-Inflammatory Effects of IOP
The elevated levels of TNF-α and TGF-β in the MC group highlight the persistent inflammatory response and pancreatic injury in CP mice. TNF-α is a key pro-inflammatory cytokine, and its increased levels indicate systemic inflammation. TGF-β is involved in fibrosis and tissue remodeling. IOP treatment significantly reduced TNF-α and TGF-β levels, demonstrating its anti-inflammatory effects. The reductions were more pronounced in the IOP-H group, suggesting that higher doses of IOP are more effective in attenuating inflammation.
Protection Against Pancreatic Damage
The elevated levels of serum lipase and pancreatic trypsin in the MC group indicate severe pancreatic damage. Lipase is a pancreatic enzyme involved in fat digestion, and its elevated levels reflect pancreatic injury. Trypsin is an enzyme that activates other digestive enzymes, and its increased levels indicate pancreatic acinar cell damage. IOP treatment significantly reduced lipase and trypsin levels, with the most substantial reductions observed in the IOP-H group. This suggests that IOP protects against pancreatic tissue damage in CP mice.
Modulation of Gut Microbiota
The study revealed significant alterations in CP mice's gut microbiota composition and diversity. The MC group exhibited reduced microbial diversity, indicating dysbiosis. IOP treatment modulated the gut microbiota, increasing the proportion of Bacteroidetes and decreasing Firmicutes. Bacteroidetes positively correlated with GSH-Px and TAOC levels, suggesting their role in antioxidative processes. Firmicutes correlated with TNF-α, TGF-β, and lipase levels, indicating their association with inflammation and pancreatic damage. These findings suggest that IOP restores gut microbiota homeostasis, contributing to its therapeutic effects in CP.
Potential Mechanisms of IOP
The therapeutic effects of IOP on CP may be attributed to its antioxidative, anti-inflammatory, and prebiotic properties. IOP's ability to increase GSH-Px and TAOC levels suggests its role in mitigating oxidative stress. Its reduction of TNF-α and TGF-β levels indicates its anti-inflammatory effects. Additionally, IOP's modulation of gut microbiota composition and diversity suggests its prebiotic effects, promoting the proliferation of beneficial bacteria and restoring microbial balance.
Conclusion
This clinical study demonstrates that Inonotus obliquus polysaccharide (IOP) effectively reduces inflammation and tissue damage in a mouse model of chronic pancreatitis. IOP's antioxidative properties increase GSH-Px and TAOC levels, mitigating oxidative stress. Its anti-inflammatory effects reduce TNF-α and TGF-β levels, attenuating inflammation. IOP also protects against pancreatic damage by reducing serum lipase and trypsin levels. Furthermore, IOP modulates gut microbiota composition and diversity, restoring microbial balance and contributing to its therapeutic effects.
This study's findings suggest that IOP can potentially be a therapeutic agent for chronic pancreatitis. Its multifaceted effects, including antioxidative, anti-inflammatory, and prebiotic properties, make it a promising candidate for further research and clinical application. Future studies should focus on elucidating the precise mechanisms of IOP's actions and evaluating its efficacy in human clinical trials.