Alteration in gut microbiota caused by time‐restricted feeding alleviate hepatic ischaemia reperfusion injury in mice

Abstract Time‐restricted feeding (TRF), that is, no caloric intake for 14‐16 hours each day leads to favourable nutritional outcomes. This study is the first to investigate TRF through a surgical perspective verifying its efficacy against liver ischaemia reperfusion (I/R) injury. We randomly assigned 100 10‐week‐old wild‐type male C57BL/6 mice into two feeding regimens: TRF and ad libitum access to food. Main outcomes were evaluated at 6, 12 and 24 hours post‐I/R surgery after 12 weeks of intervention. TRF group demonstrated minor liver injury via histological study; lower serum levels of liver enzymes, glucose and lipids; higher concentrations of free fatty acid and β‐hydroxybutyrate; decreased oxidative stress and inflammatory biomarkers; as well as less severe cell apoptosis and proliferation. Further exploration indicated better gut microenvironment and intestinal epithelial tight junction function. TRF employed its positive influence on a wide spectrum of biochemical pathways and ultimately revealed protective effect against hepatic I/R injury possibly through adjusting the gut microbiota. The results referred to a strong indication of adopting better feeding pattern for surgical patients.


| INTRODUCTION
Time restricted feeding (TRF) allows food consumption within 8-10 hours period per day, and no caloric intake is permitted during the rest 14-16 hours. No restriction is imposed on the quantity and nutritional constitution of food. Accumulated evidence has proved that TRF is a promising intervention against the worldwide trend of obesity. 1 TRF is also associated with reductions in serum total cholesterol, triglycerides (TG), glucose, insulin, interleukin 6 (IL-6), tumour necrosis factor-α (TNF-α), as well as with improvements in insulin sensitivity. 2 Feeding behaviour is predominantly dictated by our inherent circadian clock, and this timing system and other factors like food availability, social habits and convenience jointly decide the way a life entity eats. The comprehensive benefits of TRF are considered to root in its compatibility with our inborn biological clock. 3 Liver plays a critical role in the basal metabolism and progression of many diseases regarding its close relationship with food intake. 4 And the gut continuously encounters the environmental influence brought by the diet content and schedule. Therefore, the gut microbiota as a whole is responsible for maintaining a micro-homeostasis. 5 Previous studies have investigated the influence of feeding pattern on liver diseases and the hepatic injury resistance capacity. Ischaemia injury, including helium preconditioning 6 medications, 7 supplement of saturated fatty acids 8 and extracorporeal liver assist device to exchange albumin and remove endotoxin. 9 The influence of fasting on metabolic status has been proved to be modest in magnitude but broad in extent. Taken together, various indicators of systemic biochemistry dictated the boost in hepatic anti-I/R capacity caused by fasting. 10 Some studies underlined the anti-inflammatory properties of fasting, and pre-operative fasting may be an effective medical order to protect the liver against I/R injury. 11,12 Meanwhile, mechanistic studies disclosed the association between acute liver impairment with increased oxidative stress and inflammatory reaction. And fasting protected the liver from these two factors through its regulatory actions on ketone body levels. 13 However, surgical patients could hardly tolerant a malnutrition status, which largely brought down the feasibility of fasting. TRF, on the other hand, is often regarded as an alternative, or a better approach to fasting, making it promising to investigate the efficacy of this feeding pattern against liver I/R injury. was used, and drinking water was sterile filtered. We randomly assigned 100 mice into two regimens: TRF: all food consumption was restricted within an 8-10 hours period (23 PM next day 7 AM) per day, no caloric intake was allowed for the rest 14-16 hours. Ad libitum (AL): ad libitum access to food. Neither feeding pattern imposed limitation on the total amount of food the mice subjects consumed.
Dietary intervention lasted for 12 weeks before hepatic I/R operation was performed.

| Hepatic ischaemia reperfusion injury model
Partial (70%) warm ischaemia was performed as described. 14 In brief, after dissecting the portal triad, the blood supply to the left and median liver lobes was occluded with a microvascular clamp for 60 minutes, before removing the clamp to initiate reperfusion. Sham group underwent identical surgical procedures except vascular occlusion. Blood, liver and colon faecal were harvested after 6, 12 and 24 hours from post-operative mice. As the operation time is closely associated with the severity of liver injury, we finished the first surgical procedure for mice between 8 to 9 AM. Both groups went through an hour of fasting before the operation began.

| Serum biochemistry
Automatic biochemistry analyzer was used to detect the concentration of liver transaminases, blood glucose, TG, cholesterol and free fatty acids. Enzyme-linked immunosorbent assay kits were used to test the levels of TNF-α, IL-1β, IL-6, IL-10 and β-hydroxybutyrate.

| Triglycerides
Liver powder was homogenized in isopropanol, and TG concentration was measured using an enzymatic assay, and data were normalized to liver weight. Glycogen: liver samples were weighed and digested, precipitated and hydrolysed, and results were normalized to wet weight of liver. 15

| Western blotting
Western blot method was applied to detect the levels of liver NF-KB, MAPKs signal pathway protein, NLRP3 inflammatory protein complexes and LPS/TLR4 signal pathway related proteins. Liver specimens were homogenized in lysis buffer (RIPA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies at 1:1000 dilution with 5% bull serum albumin. After overnight incubation, horseradish peroxidase-conjugated second antibodies were added for 1 hour at room temperature. Glyceraldehyde 3-phosphate dehydrogenase was used for normalization.

| Histological and TUNEL analysis
Liver specimen harvest was performed at post-surgical time 6, 12 and 24 hours, and samples were fixed and embedded in paraffin.
Sections were stained with haematoxylin-eosin (H&E). Light microscopic examination of liver specimens was performed with a light microscope. Suzuki's criteria of hepatic ischaemia/reperfusion injury were used to assess the severity of liver injury in both groups. 16

Score
Congestion

| Statistical analyses
Data were analysed by Stata/MP 14.0. All normally distributed data were displayed as the mean ± SD. Pairwise comparisons between two groups were performed by the Student's t test. A value of P < 0.05 indicated statistical significance.

| Time-restricted feeding retarded weight gain
Bodyweight was monitored weekly for 12 weeks, and TRF was proved to down-regulate the weight gain. TRF mice had significant lower bodyweight, compared to the mice that had ad libitum access (AL) to food: 31.13 ± 0.76 versus 33.14 ± 0.81 g, P < 0.05, n = 50.

| TRF mitigated liver ischaemia reperfusion injury
According to the results of biochemistry profile, hepatic portal occlu-  that microbiome strongly impacts on the metabolome. 29 Fasting led to insufficient gluconeogenesis, which was recognized by hypoglycaemia and increased fatty acid released from adipose tissue, 30 in which case ketone bodies were produced in the liver and utilized in other tissues and organs. 31 Our data showed that these were true regarding TRF, and ERK, which was believed to increase liver glycogen and decrease energy expenditure in obesity, showed lower concentration in TRF mice. 32 Because the majority of the venous blood from the intestinal tract is drained into the portal circulation, the liver is hence the first organ to encounter absorbed nutrients and gut-derived bacteria and pathogens. If the gut barrier is disrupted and allows microbial products and even viable bacteria to translocate from the digestive lumen to the live, potential damage is therefore introduced to the liver. 33 In general, our data demonstrated TRF group was more inclined to ketogenic metabolic mode, one of the most evident proof was that the abundance of Firmicutes phylum in TRF mice largely exceeded that in AL mice.
And probiotics were more frequent among gut flora from TRF mice as well. This result was highly in accordance with the fact that the injure brought by I/R operation was greatly shaded by TRF. It was believed that intestinal mucosa barrier function mediated the effect of gut microbiome on the body. 34 And the intestinal epithelial tight junctions are charged with complex task of supporting transportation of nutrients while providing a barrier to microbial translocation, making the intestinal tract a unique organ where the permeability regulation is worth exploring, 35 and even became a therapeutic target for a variety of diseases. 36 Cytoplasmic adaptor proteins such as zo-1 and some other protein components jointly seal the space F I G U R E 5 Gut microbiota. (A) Showed top 10 bacteria under phylum, class, order and family levels. Faeces samples were harvested at 6, 12 and 24 h after hepatic portal occlusion surgery, and controls were collected from sham operation group, n = 6. (B) Taxonomy tree revealing the relative abundance of the most frequent species detected. Different faeces samples collected at different timing from either AL or TRF group were indicated by different colours, and the sizes of circles represented the proportion of the corresponding micro-organism, n = 6. SCAL: surgical control ad libitum; S6hAL: surgical 6 h ad libitum; S12hAL: surgical 12 h ad libitum; S24hAL: surgical 24 h ad libitum; SCTRF: surgical control time-restricted feeding; S6hTRF: surgical 6 h time-restricted feeding; S12hTRF: surgical 12 h time-restricted feeding; S24hTRF: surgical 24 h time-restricted feeding between cell-cell adhesion. 37 The level of zo-1 in TRF group was higher than that of the AL group, reflecting a more integrated barrier function under this feeding regimen. 38 Previous studies have profoundly proved that gut microbiota was closely related to a wide spectrum of diseases including inflammatory bowel diseases, allergies, metabolic disorders and liver disease. 39 Yet the correlation of gut flora and IR was not investigated before, which arose an insight into this feeding regimen and surgical intervention.

| CONCLUSION
Time-restricted feeding improved intestinal microenvironment and strengthened the intestinal epithelial tight junction, which provided favourable conditions leading to better energy metabolic status, inhibited inflammatory reaction, alleviated oxidative stress and suppressed hepatocytes apoptosis and proliferation. All these factors jointly contributed to an enhanced tolerance against I/R injury, indicating that adopting TRF regimen is highly recommended in patients scheduled for liver surgeries.

This study was supported by CAMS Innovation Fund for Medical
Sciences (2016-I2M-1-001) and National High-tech R&D Program