TXF dose-dependently increased glucose uptake (GU) in L6 myotubes under the condition of insulin absence. This increase in GU was partially, but significantly canceled by TXF treatment in combination with either LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), which phosphorylates protein kinase B (Akt) or Compound C, an inhibitor of 5’-adenosine monophosphate-activated protein kinase (AMPK). Furthermore, TXF was demonstrated to activate (=phosphorylate) both Akt and AMPK, and promote glucose transporter 4 (GLUT4) translocation to the plasma membrane from cytosol of L6 myotubes via both PI3K/Akt and AMPK signaling pathways. Based on these in vitro findings, we conducted an in vivo experiment in KK-Ay/Ta mice with hyperglycemia and hyperuricemia. Fasting plasma glucose, insulin, uric acid levels and an index of insulin resistance (HOMA-IR) increased significantly in the T2D model mice compared with normal ones. Such rises in the T2D state were significantly suppressed by oral administration of TXF for four weeks. These results suggest that TXF is a potent antihyperglycemic and antihyperuricemic phytochemical in the T2D state.Muscle is the largest tissue in our body and plays an important role in glucose homeostasis and hence diabetes. In the present study, we examined the effects of taxifolin (TXF) on glucose metabolism in cultured L6 muscle cells (myotubes) and in type 2 diabetic (T2D) model KK-A Aspalatsistance ,13,14.Taxifolin (TXF) or dihydroquercetin is widely found in various plants  includiny/Ta mice were significantly higher than those in normal mice. In addition, plasma uric acid levels showed significant and positive correlations with plasma glucose, insulin, HOMA-IR and triglyceride levels [Meanwhile, Zhu et al. demonstrated that high uric acid (UA) directly inhibits insulin signaling and induces insulin resistance . Adachi e levels . TXF hase levels .y/Ta mice in vivo. Results demonstrate that TXF is a potent anti-hyperglycemic and anti-hyperuricemic phytochemical.We have recently found a possibility that TXF promotes GU in cultured L6 myotubes under the condition of insulin absence. In the present study, we confirmed this possibility by investigating the dose-responses and modes of action of TXF on GU in L6 myotubes in vitro and examined the effects of TXF on hyperuricemia as well as hyperglycemia in T2D model KK-A®, CRL-1458, Lot No. 61875400. Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium . The medium was supplemented with 10% (v/v) fetal bovine serum (FBS) , L-glutamine , 0.06% sodium bicarbonate , and penicillin–streptomycin mixed solution  under an atmosphere of 5% CO2/95% humidified air at 37 °C. Culture of myocytes and glucose uptake assay and biochemical analyses were conducted essentially as described previously [4 cells/well) were subcultured into Multicell Plate for Cell/Tissue Culture 24F with a lid  and grown to 90% confluency for three days in 0.4 mL of 10% FBS/DMEM. Next, the cells were cultured and differentiated to myotubes in 2% FBS/DMEM for one week. The fresh medium was replaced every two days. Differentiated L6 myotubes were finally incubated in filter-sterilized Krebs–Henseleit–HEPES buffer (KHH buffer)  containing 0.1% bovine serum albumin , 10 mM HEPES, and 2 mM sodium pyruvate) for 2 h. TXF for treatment was dissolved in dimethyl sulfoxide  and diluted in KHH buffer containing 11 mM (198 mg/dL) glucose . Similarly, KHH buffer containing 11 mM glucose for the control (0 µM TXF) was prepared with 0.1% DMSO alone. The myotubes were then incubated for 4 h in KHH buffer containing glucose without or with 50–200 µM taxifolin (TXF)  in the dose-response study. Likewise, the myotubes were incubated for 4 h without or with 25 µM LY294002  as an Akt inhibitor or 10 µM Compound C (dorsomorphin)  as an AMPK inhibitor, in the absence or presence of 100 µM TXF. An Akt activator, human recombinant insulin  and an AMPK activator, AICAR  were treated to the cells as the positive control. The differences in the glucose concentrations in the KHH buffer before and after culture were determined with a Glucose CⅡ-Test Kit  and a microplate reader SPARK™ 10M  at 505 nm. The amount of glucose consumed was calculated. Similar experiments were conducted at least three times in myotubes differentiated from myoblasts with different passage numbers to confirm the reproducibility of results.L6 myoblasts were provided from American Type Culture Collection  were subcultured into Nunc EasYDishes 60 mm type  and grown to 90% confluency for three days in 3 mL of 10% FBS/DMEM. Then, the cells were cultured to form myotubes in 2% FBS/DMEM for one week. The fresh medium was replaced every two days. After L6 myotubes were kept in KHH buffer for 2 h, the myotubes were then incubated in KHH buffer containing 11 mM glucose without or with 100 µM TXF suspended in 0.1% DMSO for 15–60 min. In the same way, KHH buffer containing 11 mM glucose for control (0 µM TXF) was prepared with 0.1% DMSO alone. To extract protein, the cells were collected from the dishes into RIPA buffer with Protease Inhibitor Cocktail  containing 1% (v/v) Phosphatase Inhibitor Cocktail  and sonicated for 5 s on ice. The cell lysate was centrifuged at 10,000× g for 10 min at 4 °C. The supernatant was collected as a protein extract. The protein extracts for Akt, AMPK, and their phosphorylation detections were mixed at 1:1 with 4% SDS, incubated at 100 °C for 3 min and stored at −80 °C until use.L6 myoblasts (5 × 10v/v) IGEPAL CA-630 , and homogenized with a 27G needle . Each homogenate was centrifuged at 1000× g for 10 min at 4 °C. The supernatant was placed on ice for 1 h and centrifuged at 16,000× g for 20 min at 4 °C. The obtained supernatant was collected as the post-plasma membrane fraction. The precipitate was suspended in IGEPAL CA-630-free buffer A, and centrifuged at 1000× g for 10 min at 4 °C. The precipitate obtained was resuspended in buffer A containing 1.0% (v/v) IGEPAL CA-630, stood on ice for 1 h after mixing using the needle, and centrifuged at 16,000× g for 20 min at 4 °C. The supernatant was collected as the plasma membrane fraction [The plasma membrane and post-plasma membrane fractions were obtained by the methods described by Nishiumi and Ashida  with slifraction . The pro® PVDF membranes for protein blotting  . The membranes were incubated in 5% (w/v) BSA/Tris buffered saline with Tween 20 (BSA/TBST) at room temperature for 1 h. After the incubation, the membranes were incubated with primary antibodies: p-AMPK (Thr 172) rabbit antibody , p-Akt (Ser 473) rabbit antibody , AMPK rabbit antibody , Akt rabbit antibody , GLUT4 (1F8) mouse antibody , and Na+/K+-ATPase rabbit antibody  for 16 h at 4 °C. The membranes were then washed three times with TBST and incubated with secondary antibodies: anti-rabbit IgG HRP linked whole antibody from donkey  or anti-mouse IgG HRP linked whole antibody from sheep , for 1 h at room temperature. After washing the membrane three times with TBST, the blots on the membrane were developed by Amersham™ ECL™ Western Blotting Detection Reagent  and analyzed with ChemiDoc™ XRS+ with Image Lab™ Software . These processes were conducted essentially based on the previous report [The protein concentrations of each supernatant were evaluated using Pierce™ BCA Protein Assay Kit  and a microplate reader  at 562 nm. Equal amounts of protein (15 µg/lane) were loaded onto 10% polyacrylamide gels, separated by electrophoresis (100V), and transferred to Immuno-Bloty/TaJcl (KK-Ay/Ta) mice  and C57BL/6JJcl (C57BL/6J) mice  were purchased from CLEA Japan Inc., Tokyo, Japan. KK-Ay/Ta mice were adopted as type 2 diabetes (T2D) model animals and C57BL/6J mice as non-diabetic normal ones, since the KK-Ay/Ta strain is derived from C57BL/6J strain. The mice were housed in an individual cage, maintained in a controlled environmental room . All mice were supplied with a standard mouse chow CRF-1  and water ad libitum for the entire duration of the study. After acclimation for one week, the KK-Ay/Ta mice (five weeks old) were divided into two groups: diabetic control group  and TXF orally administered group at 30 mg/kg body weight/day , with similar blood glucose levels and body weights (0 week). TXF was suspended in 0.5% (w/v) carboxymethyl cellulose sodium salt  solution and orally given by a sonde once a day in the morning for four weeks at a dose of 0.9 mg/0.3 mL/30 g body weight/day = 30 mg/10 mL/kg body weight/day. The non-diabetic normal group  and diabetic control group (CNT) were given 0.5% (w/v) CMC solution alone for four weeks. The animal experiment described here was carried out in accordance with the guideline for the Animal Experiments of Utsunomiya University Animal Research Committee and was approved by this committee .KK-Aw/v) trichloroacetic acid (TCA)  aqueous solution (25 µL) was added and kept on ice. The mixture was centrifuged at 12,000× g for 5 min at 4 °C. The supernatant was collected for determination of the blood glucose levels with a Glucose CⅡ-Test Kit  and a microplate reader SPARK™ 10M  at 505 nm, in every week for four weeks. To evaluate the effect of TXF on blood glucose levels, the blood from the tail vein of mouse was collected once a week for four weeks. The mice were fasted for 90 min from the oral administration at 9:00, but allowed free access to water until blood collection. Blood (5 µL) was collected from tail vein, bursted in 20 µL distilled water, then 20% , Morinaga Ultra Sensitive Mouse Insulin ELISA Kit , Triglyceride E-Test Kit , Cholesterol E-Test Kit , Uric Acid C-Test Kit , and Mouse Adiponectin/Acrp30 Quantikine® ELISA Kit , respectively. These absorbances were read at 450 nm (insulin and adiponectin), 505 nm (glucose), 555 nm (uric acid), and 600 nm  using a microplate reader SPARK™ 10M . HOMA-IR, an index of insulin resistance, was calculated from plasma glucose and insulin concentrations, as described previously [At 270 min  fasting after the final blood collection from the tail vein at the fourth week, whole blood was drawn from the inferior vena cava of the mice (nine weeks old) anesthetized with isoflurane  and then placed into a heparin-coated tube on ice. The whole blood was centrifuged at 1200× eviously ,26. p < 0.05 and ** p < 0.01. All statistical analyses were interpreted using the Prism 6 program for Mac OS X .All data are represented as the mean values ± SEM. Data on the glucose uptake and the protein expression in L6 myotubes were analyzed by one-way ANOVA with post-hoc Tukey–Kramer’s multiple comparisons test. Data on levels of blood glucose and plasma components were analyzed by one-way ANOVA with post-hoc Dunnett’s multiple comparisons test. Statistical significance was defined as * First, we investigated the effect of TXF A on GU fNext, we tried to confirm phosphorylation (=activation) of Akt (p-Akt) and AMPK (p-AMPK) in L6 myotubes by western blot analyses. The ratios of p-Akt to total Akt (p-Akt/Akt) A and p-A+/K+-ATPase was adopted as muscle plasma membrane (PM) marker. Insulin and AICAR were used as positive controls for the Akt and AMPK pathways, respectively. As shown in +/K+-ATPase was recognized in the PM fraction, while that in the Post PM fraction was extremely faint. In the PM fraction, TXF treatment significantly raised GLUT4/Na+/K+-ATPase ratio three times as high as that of no TXF treatment, and this increase in GLUT4 translocation to the plasma membrane was considered to promote GU in L6 myotubes.As TXF was found to induce phosphorylation (=activation) of both Akt and AMPK in L6 myotubes, we finally confirmed the translocation of GLUT4 to plasma membrane from cytosol of L6 myotubes by western blot analysis after 30 min exposure of TXF to L6 myotubes . Na+/K+-y/Ta mice. As shown in From the in vitro results obtained from cultured L6 myotubes, TXF was suggested to be capable of promoting glucose transfer from the blood stream into muscle tissues via GLUT4, leading to the hypoglycemic effect in vivo. Therefore, we decided to examine whether or not TXF has a hypoglycemic effect in T2D model KK-Ay/Ta mice (TXF).p < 0.1) by oral administration of taxifolin (TXF) (y/Ta mice as previously reported [y/Ta mice (CNT) in comparison with normal ones (NOR), and TXF administration exerted no influence on this reduction (TXF) (in (TXF) A,B. The reported , and TXFreported , signifion (TXF) D.In the present studies, TXF was demonstrated to dose-dependently increase GU in cultured L6 myotubes via both PI3K/Akt and AMPK signaling pathways such as resveratrol . The acty/Ta mice  AMPK (p-AMPK) in gastrocnemius muscle tissues, and to suppress the increase in blood glucose levels in vivo . In the y/Ta mice compared with normal ones, and TXF exerted no influence on the reduction  . Since Khropathy ,35, it sy/Ta mice. High uric acid (UA) is demonstrated to directly inhibit insulin signaling by insulin receptor modification and induce insulin resistance [y/Ta mice, and TXF was found to alleviate these symptoms. TXF was reported to suppress purine body-induced hyperuricemia by inhibiting hepatic xanthine oxidase, a key enzyme for UA production [y/Ta mice from the aspects of production and excretion of UA [Another important finding in the present study is the anti-hyperuricemic effect of TXF in T2D model KK-Asistance . As alresistance , we couloduction . Studieson of UA . In the present study, TXF was suggested to upregulate both PI3K/Akt and AMPK signaling pathways in L6 myotubes. However, it is necessary and interesting to perform combined treatments of LY294002 and Compound C to confirm that their effect is additive and that the pathways work independently of each other. Since a crosstalk between the Akt and AMPK pathways cannot be excluded ,38, it wy/Ta mice in the present study (Luo et al. indicated that the development of atherosclerosis and inflammation is promoted by uric acid in vivo. Moreover, the lowering of uric acid levels attenuated inflammation via the activation of the AMPK pathway . Kimura nt study C. These As genotoxicity  and muta


Background: Spinal cord injury (SCI) is characterized by autonomic dysreflexia, chronic pain, sensory and motor deficits. Resveratrol has shown potential neuroprotective function in several neurodegenerative diseases’ models. However, if resveratrol could improve the function recovery after SCI and the further mechanism have not been investigated.Methods: SCI rat model was established through laminectomy at lamina T9-10 aseptically. Basso, beattie and bresnahan (BBB) and inclined plane score, sensory recovery, spinal cord content, and inflammatory factors were measured. The levels of GAP43, NF421, GFAP, Bax, Bcl-2 and caspase-3 were measured using immunohistochemical staining. Tunel staining was applied to detect apoptosis level.Results: Resveratrol significantly improved the function recovery, promoted axonal regeneration, suppressed apoptosis after SCI. The activation of Wnt/β-catenin signaling pathway was achieved by resveratrol. XAV939 significantly reversed the influence of resveratrol on function recovery, axonal regeneration, apoptosis after SCI.Conclusions: Resveratrol could promote the function recovery and axonal regeneration, improve histological damage, inhibit apoptosis level after SCI through regulating Wnt/β-catenin signaling pathway. This research expanded the regulatory mechanism of resveratrol in SCI injury. Spinal cord injury (SCI) refers to the spinal cord injury caused by external direct or indirect factors, which often leads to partial or total loss of sensory and motor functions below the injury level , 2. EpidRecent years, the function of resveratrol has been widely reported, and resveratrol is widely existed in grape, pine, Polygonum cuspidatum, cassia seed, peanut and other natural plants or fruits , 8. SomeWnts containing wnt1, wnt3, and wnt5, are known as glycoproteins. Wnts have been proved to be closely related with cell proliferation, invasion, migration, and apoptosis in several pathological processes including tumor, myocardial ischemia reperfusion injury, and SCI –13. PrevIn this study, the SCI animal model was established to investigate the regulatory role of resveratrol in function recovery after SCI injury. We found that resveratrol could promote the function recovery and axonal regeneration, improve histological damage, inhibit apoptosis level after SCI through regulating Wnt/β-catenin signaling pathway. The novel findings of this research expand the regulatory mechanism of resveratrol in SCI injury, and might provide a potential therapeutic thought for SCI.Sprague-Dawley rats  were used in this study. Animals were kept in the pathogen free animal center of the 900th hospital. The environment was maintained with 12 h light-dark cycle, 23-27° C. All experiment protocols have been approved by the experimental animal ethics committee of the 900th hospital, Joint Logistics Support Force . The animals were divided into the following groups randomly: Sham, SCI, SCI+resveratrol, SCI+resveratrol+XAV939. The animals in the sham group were treated with laminectomy. In the group SCI+resveratrol, 30 mg/kg of resveratrol was injected intraperitoneally daily for the first 14 days after SCI. In the group SCI+resveratrol+XAV939, 0.5 mg/kg of XAV939 and 30 mg/kg of resveratrol were injected intraperitoneally daily for the first 14 days after SCI , 16. SamAnimals were anesthetized by intraperitoneal injection with xylazine (10 mg/kg) and ketamine (100 mg/kg) . After a2. After trypsin digestion, the cells were used for apoptosis measurement. The H2O2 treated cell model was established through culturing cell with H2O2 (100 μM) for 4 h at 37° C with 5% CO2. In the group H2O2+resveratrol, cells were incubated with resveratrol (10 μM) for 6 h after H2O2 treatment at 37° C with 5% CO2. In the group H2O2+resveratrol+XAV939, cells were incubated with resveratrol (10 μM) and XAV939 (5 μM) for 6 h after H2O2 treatment. In the group H2O2+resveratrol+iCRT3, cells were incubated with resveratrol (10 μM) and iCRT3 (5 μM) for 6 h after H2O2 treatment.Murine microglia BV2 cells were purchased from Chinese Academy of Science . Cells were cultured in DMEM medium containing 10% fetal bovine serum  at 37° C with 5% CO4) were plated into the 96-well plate. The H2O2 treated cell model was established as described above. The cells in the group H2O2+resveratrol were cultivated with resveratrol (10 μM) for 6 h at 37° C with 5% CO2. The cells in the group H2O2+resveratrol+XAV939 were cultivated with resveratrol (10 μM) and XAV939 (5 μM) for 6 h at 37° C with 5% CO2. The cells in the group control were cultivated with same amount of PBS. Then, 20 μL CCK-8 regent was added to each well. After 1 h incubation, OD at 492 nm was measured.Cells  and ketamine (100 mg/kg), and spinal cords were resected. The tissues were fixed using 4% paraformaldehyde for 48 h. Then, the tissues were dehydrated using 20% sucrose. The 10 μm crosswise sections were prepared for HE and immunohistochemical staining.The tissues were stained using hematoxylin for 45 s, and rinsed in distilled water for 10 s. HCl/95% alcohol (1:100) was used for differentiation (5 s). After washing using water for 30 min, tissues were stained with eosin for 15 s. Then, sections were washed with water for 10 min, and mounted using neutral gum after dehydration.The spinal cord tissues were minced firstly, and homogenized using lysis buffer. After centrifugation (30 min at 4° C), the supernatant samples (30 μg) were subjected to SDS-PAGE, and transferred to a PVDF membrane . After blocking with TBST containing 10% non-fat milk for 1 h, the membrane was incubated with primary antibodies at 4° C for 12 h. The primary antibodies information was listed as follows. Wnt3a , Wnt2b , Wnt7a , Abcam, UK), phospho-GSK-3β , β-catenin . The membrane was then incubated with secondary antibodies  at 4° C for 2 h. Then, proteins were detected using an enhanced chemiluminescence detection kit , and protein bands was analyzed with ImageJ software.To measure the protein expression of β-catenin in the nuclear, Nuclear Complex Co-IP Kit  was used to exact nuclear firstly according to the instruction.-ΔΔCT method.Total RNA was extracted using the RNA extraction kit . ReverTra Ace qPCR RT Master Mix kit  was used to synthesize cDNA. Then, ABI5500 system (Strata-gene) was use to perform qPCR. The reaction system was set as follows: 95° C (40 s), 40 cycles of 95° C (20 s), 60° C (40 s), 72° C (50 s). Primer sequences used listed as follows: Wnt3a ; β-catenin ; GSK-3β ; GAPDH . The gene expression was analyzed using 22. After different treatments as described in 2.2, the cells were digested using trypsin. After centrifugation for 5 min at 2500 rpm, the cells were collected and suspended with PBS. Then, propidium iodide (10 μL) and Annexin V-FITC (10 μL) were added to cells, and incubated for 20 min in the dark at room temperature. Finally, cell apoptosis was measured with flow cytometry.Cells were firstly cultured at 37° C and 5% COThe tissues were treated with 0.01 M citric acid for antigen retrieval. After washing with PBS, the slides were blocked with TBST containing 10 goat serum for 1 h. Then, the tissues were incubated with primary antibodies overnight at 4° C. The primary antibodies and dilution times were listed as follows. GAP43 , NF421 , GFAP , Bax , Cleaved Caspase-3 , Bcl-2 . Then, the sections were washed using PBS and incubated with related secondary antibodies for 2 h at room temperature. The secondary antibodies and dilution times were listed as follows. Goat anti-rabbit lgG  and Goat anti-mouse lgG . All sections were mounted and observed under a microscope . Image J software was used to quantify and normalize images.2. After washing with PBS, the sections were mounted and observed using a microscope.After antigen retrieval and blocking as described in the 2.9, the sections were incubated with primary antibodies overnight at 4° C. The sections were incubated with 15 μg/mL proteinase K (30 min), 0.2% hydrogen peroxide (40 min), and 0.1% Triton X-100 (5 min) successively. After washing with PBS, the tissues were blocked at 4° C for 4 h with 5% goat serum. After removing the serum, the sections were incubated with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) color liquid in the dark for 30 min at 37° C with 5% CO2 area for free movement. The behavior of hind limbs, tail, and trunk of animals were recorded. The BBB score was evaluated by three different experimenters who were blinded to the grouping. Von Frey filament test was applied to evaluate sensory recovery. Filaments with a gradient force were used to the paws to induce nociceptive responses . The withdrawal threshold value is defined as the minimal force inducing positive responses.The BBB score was measured as described previously . The aniAnimals were put on the inclined plane firstly. Then, the plane was then raised to the maximum angle, and the maximum vertical axis of the inclined plate was measured. Each rat was tested for 5 times.α (#PT516) with related ELISA assay kits .The spinal cord tissues were minced firstly, and homogenized using lysis buffer. After centrifugation (30 min at 4° C), the supernatant samples were used to measure the expression of IL-6 (#PI328), IL-1β (#PI303), and TNF-The spinal cords were dried for 72 h at −80° C, and this was used to measure dry weight. Water content in spinal cords was calculated the formula: (wet weight − dry weight)/wet weight × 100%.Data was shown as mean ±SD, and analyzed with SPSS software . An unpaired 2-tailed Student’s t-test was applied to compare the data of two groups. p <0.05 was believed to be statistically different.Data supporting this study has been presented in the manuscript.The BBB scores were evaluated after operation at different time points. The BBB scores dropped to zero 1 day after surgery in the group SCI and SCI+Resveratrol , and it Histological changes were evaluated using HE staining. In the group sham, clear and symmetric axons, myelin, nerve fibers could be observed. Nerve cells could be observed in the gray matter . Howeverin vitro. H2O2 was used to establish cell apoptosis model in vitro. The remarkable increased apoptosis level induced by H2O2 was suppressed by resveratrol (2O2 was promoted by resveratrol (The apoptosis level in the tissue was detected using TUNEL staining. The apoptosis level in the group SCI was remarkably elevated compared group sham, but it was significantly suppressed after resveratrol treatment . Meanwhiveratrol , 3H. Meaveratrol .The protein and mRNA expression of Wnt3a, GSK-3β, and β-catenin in the tissues were measured. The protein and mRNA levels of Wnt3a and β-catenin were significantly inhibited after SCI treatment –4C compaXAV939, the inhibitor of Wnt/β-catenin signaling pathway, was used to investigate the regulation of resveratrol on Wnt/β-catenin signaling pathway. Significant higher levels of BBB score, inclined plane score, sensory recovery, and weight gaining caused by resveratrol were markedly decreased by XAV939 –5D. In aWe found that the influence of SCI on histological changes, the expression of GAP43, NF421, and GFAP could be remarkably reversed by resveratrol . HoweverSimilar to previous findings, the apoptosis in the tissues  and cellin vitro. iCRT remarkably suppressed the protein (Another inhibitor of Wnt/β-catenin signaling, iCRT, was used to investigate the role of Wnt/β-catenin signaling in apoptosis  protein , 8B and  protein  levels o protein , 8E and  protein  were rev protein , 8H.SCI always causes irreversible physical function injury, and it is still a global challenge. The current treatment drug of SCI, methylprednisolone, has been proved to exert some complications such as pulmonary embolism and infection , 20. TheThe difficulty of SCI treatment is to promote the injured axons to regenerate and link with target cells, so as to achieve nerve reinnervation and functional recovery . Axon reResveratrol has been proved to be beneficial in a broad type of pathological conditions including tumor, myocardial ischemia reperfusion injury, and oxidative stress . PreviouGAP43, also known as neurodulin, is an axon membrane protein. It is a nerve specific protein, which is involved in nerve extracellular growth, synaptic development and regeneration. GAP-43 is highly expressed during neuronal development and regeneration. It can mediate axon extension . NF421 iApoptosis is closely related with the process of tissue destruction and cell stress, and the level of apoptosis is highly elevated after SCI. Apoptosis could cause nerve impairment and further induce sensory and motor damage . In addiIn this study, we demonstrated that resveratrol could significantly improve neurological function recovery, promote axonal regeneration, and suppress apoptosis after SCI injury through activating Wnt/β-catenin signaling pathway. This study unfolds the novel regulatory mechanism of resveratrol in improving neurological recovery. Meanwhile, this study might provide a potential therapeutic strategy for the treatment of SCI through resveratrol promoting axonal regeneration and activating Wnt/β-catenin signaling pathway.Supplementary Figures


IntroductionPrimary hyperparathyroidism (PHPT) is an endocrinological disorder associated with increased systemic inflammation. This study aimed to examine the changes in the neutrophil/lymphocyte ratio (NLR), serum parathormone, serum corrected calcium, serum phosphate, and white blood cell (WBC) count in patients with PHPT before and after parathyroidectomy.MethodsA total of 37 patients who underwent successful parathyroidectomy for PHPT were included in the study. NLR, serum parathormone, serum corrected calcium, serum phosphate, and WBC count were compared before parathyroidectomy and at the sixth postoperative month.ResultsThe difference in the NLR, serum parathormone, serum corrected calcium, and serum phosphate values before and after parathyroidectomy was statistically significant , but there was no significant difference in the WBC count (p=0.314). The correlation analysis performed before parathyroidectomy revealed a significant positive correlation between NLR and serum parathormone , serum corrected calcium  and WBC count , and a significant negative correlation between NLR and serum phosphate (r=-0.331).ConclusionIn patients with PHPT, successful parathyroidectomy results in a decrease in NLR. Increased systemic inflammation in patients with PHPT can be reduced following parathyroidectomy. Primary hyperparathyroidism (PHPT) is an endocrine disease characterized by the excessive autonomic production of parathormone (PTH), increased serum PTH and calcium (Ca) levels as a result of the impaired regulation of Ca metabolism, or an inappropriate increase of Ca and PTH -3. PTH pStudies have found that there is a relationship between a high serum PTH level and various inflammatory markers  . ConsideStudies show a complex interaction between inflammation and changes in PTH levels. High PTH levels have been shown in septic patients . PTH proVarious biochemical and hematological markers are available to assess systemic inflammation, but some are costly to use. It is important that the marker to be used as an indicator of inflammation is inexpensive and easily available . The neuThis study aimed to examine the pre- and post-PTX values of NLR, which is an indicator of systemic inflammation in patients with PHPT, and compare its relationship with biochemical variables.This research has received approval from the Adiyaman University Clinical Research Ethics Committee  and included 37 patients who underwent successful PTX due to PHPT between June 2017 and June 2020 in the general surgery clinic of Adiyaman University Training and Research Hospital. The data of these patients were retrospectively analysed. Age, gender, serum Ca, serum PTH, serum albumin, serum phosphorus, white blood cell (WBC) count, neutrophil, and lymphocyte values were noted before PTX and the sixth postoperative month. The formula [(4-Albumin (gr/dl)) * 0.8 + serum Ca (mg/dl)] was used to calculate the corrected Ca value. NLR was obtained by simply dividing the neutrophil count by the lymphocyte count.According to the 4th International Workshop guidelines, a multidisciplinary council made the decision to recommend PTX in patients with serum Ca level being 1 mg/dl higher than the upper limit of the normal range, those with decreased bone mineral density , those with renal failure (glomerular filtration rate < 60 ml/min), nephrolithiasis cases, patients aged under 50 years, and those that did not want to or could not undergo long-term medical treatment .Hypercalcemia that persisted after PTX or recurred within six months was accepted as persistent hyperparathyroidism, and cases that developed hypercalcemia after six months or later were considered as recurrent hyperparathyroidism. Patients with persistent and recurrent hyperparathyroidism, malignancy, diabetes mellitus, hyperthyroidism, multiple endocrine neoplasia, and systemic infections were excluded from the study.Statistical analysis was carried out using the Statistical Package for the Social Sciences . Numerical variables were stated as the mean and standard deviation of minimum and maximum values, while categorical variables were obtained as numbers and percentages. The distribution of data was evaluated using the Shapiro-Wilk test. Pre- and post-PTX variables were analysed using the paired Student's t-test. Pearson’s correlation analysis was used to determine possible relationships between the variables. A p-value of less than 0.05 was considered statistically significant.This retrospective study included 37 patients with PHPT who underwent successful PTX. Thirty-three (89.2%) of the patients were female, and four (10.8%) were male. The median age was 54  years. There was a weak inverse correlation between the age of the patients and their preoperative serum PTH, serum phosphate, WBC count and NLR values , but it was not at a statistically significant level. There was a weak correlation between the patients' age and the serum corrected Ca values (r = 0.243), and this was also not statistically significant.The mean preoperative serum corrected Ca value of the patients was 13.69 ± 0.65 mg/dl , and the mean postoperative corrected mean Ca value was 9.19 ± 0.52 mg/dl (min: 8.18-max: 9.9). There was a statistically significant difference between the preoperative and postoperative serum corrected Ca values (p < 0.001). The mean preoperative and postoperative serum PTH values of the patients were 214.61 ± 151.87 pg/ml (min: 74.1-max: 843.0) and 47.35 ± 17.30 pg/ml (min: 24.7-max: 98.6), respectively, indicating a statistically significant difference (p < 0.001). The mean serum phosphate values of the patients were 2.39 ± 0.44 mg/dl (min: 1.60-max: 3.20) preoperatively and 3.30 ± 0.38 mg/dl (min: 2.50-max: 4.20) postoperatively, with a statistically significant difference (p < 0.001). The mean preoperative WBC count was 7.25 ± 1.03 (*103/µl) (min: 5.21-max: 8.93) while the mean postoperative WBC count was 7.50 ± 1.18 (*103/µl) (min: 5.30-max: 9.27), revealing no statistically significant difference (p = 0.314). The mean preoperative and postoperative NLR values of the patients were 2.28 ± 0.65 (min: 1.24-max: 3.52) and 2.20 ± 0.59 (min: 1.24-max: 3.52), respectively. There was a statistically significant difference between the preoperative and postoperative NLR values (p = 0.019) , a weak non-significant inverse correlation with serum phosphate , a weak non-significant correlation with the WBC count (r = 0.231), and a moderate significant positive correlation with NLR . The preoperative serum corrected Ca had a weak significant inverse correlation with serum phosphate , a weak non-significant correlation with the WBC count (r = 0.241), and a weak significant correlation with NLR . The preoperative serum phosphate had a non-significant inverse correlation with the WBC count (r = -0.307) and a weak significant inverse correlation with NLR . Lastly, there was a moderate significant correlation between the preoperative WBC count and NLR   . Lam et As expected, in patients with PHPT, the serum PTH and corrected Ca levels are higher, and the serum phosphate values are lower than the standard ranges . In a stOur study's most important limitations concern its retrospective nature, small sample size, and the absence of a control group. Prospective studies to be conducted with larger patient populations may provide more meaningful results.Our study observed that NRL, a systemic inflammation marker, decreased after PTX in patients with PHPT. The data obtained from the study show that PTX reduces the risks of increased systemic inflammation, as well as providing treatment for patients with PHPT.


Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies. These are non-enveloped viruses consisting approximately 7.5 kb positive-sense, single-stranded RNA genome. They possess an icosahedral capsid consisting of 60 subunits with four structural viral proteins (VPs): VP1 to VP4. The coding region encodes a single polyprotein flanked by non-translated regions at both the 5′ and 3′ ends. Group A and group B coxsackieviruses belong to the genus Enterovirus, and several serotypes have been identified: 23 in group A and six in group B. Although some syndromes are caused only by group A coxsackieviruses, diseases like myocarditis and pancreatitis are caused mainly by group B coxsackieviruses2.Enteroviral infections are common in humans worldwide 3, in addition to causing acute pancreatitis4 and meningitis in those affected5. CVB-reactive antibodies have been detected in ~ 50% of dilated cardiomyopathy (DCM) patients, while enterovirus genomic material has been detected in up to 70%9, suggesting that CVB can be an important trigger of myocarditis/DCM and possibly involve autoimmune responses to cardiac antigens. Indeed, by using major histocompatibility complex (MHC) class II dextramers, we had previously demonstrated that A/J mice infected with CVB3 generate pathogenic myosin-specific T cells, suggesting that autoimmunity is an important sequalae of viral myocarditis10. While this observation raises concerns about the use of wild type (wt) viruses as vaccine candidates, successful derivation of altered viruses as vaccine candidates, free of virulence attributes and side effects like cardiac autoimmunity, is a challenging task in vaccine research.Within group B coxsackieviruses, coxsackievirus B (CVB)3 is commonly implicated in the causation of myocarditis11. Using this virus as a backbone, we created a series of mutant viruses and identified one virus, termed Mutant (Mt) 10, that offered complete protection from both myocarditis and pancreatitis in challenge studies, suggesting that the Mt 10 virus could be used as a vaccine candidate. Mechanistically, the vaccine responses were accompanied by induction of both virus-neutralizing antibodies (nABs) and antigen-specific-T cell responses, with no evidence of autoimmunity or cardiac injury.In our efforts to analyze critical viral regions required for CVB3 to enter the target cells through coxsackievirus-adenovirus receptor (CAR) interaction, we noted one region corresponding to VP1 771–790 that participates in CAR binding. Recently, we created an infectious clone of CVB3 (pBRCVB3) whose ability to induce myocarditis was significantly attenuated, while occurrence of pancreatitis remained unaltered11. Using this virus as the backbone, we sought to create attenuated mutant viruses that would lose their ability to induce both myocarditis and pancreatitis, thus leading us to identify one mutant virus possessing a mutation in the CAR-binding region as a vaccine candidate in the prevention of CVB3 infection.By generating a full-length infectious clone of the CVB3 genome (pBRCVB3), we had previously reported that the pBRCVB3 virus induces mainly pancreatitis, but its myocarditis-inducing ability was severely impaired12. We targeted VP1, one of the four structural proteins of CVB3 that has been identified as an immunogenic protein in other enteroviruses15. Using an overlapping peptide library, we identified three epitopes—VP1 681–700, VP1 721–740, and VP1 771–790—that induce antigen-specific, CD4 T cell responses as evaluated by MHC class II dextramers/tetramers12. Next, we took advantage of crystal structure data (PDB: 1COV)16 and, using the Discovery studio visualizer software, retrieved the footprints of the putative binding regions of CVB3 and CAR to determine the locations of viral epitopes. Using the cryo-electron microscopic model17, we mapped the VP1 to VP4 proteins as illustrated in the ribbon and surface models ; N, threonine (T), and NN; shown as balls] were exposed within VP1 771–790 , whereas those infected with pBRCVB3 had a similar decline in relation to saline recipients (p ≤ 0.0001), but it occurred progressively as expected11. In contrast, animals infected with the mutant viruses remained clinically healthy and did not lose body weight compared to wt CVB3 or pBRCVB3 groups (p ≤ 0.0001), except for mice infected with Mt 3. We then compared mortalities between groups, and, expectedly, all animals infected with the wt CVB3 died by day 9 p.i (100%), as opposed to 20% (2/10) of those infected with pBRCVB3 virus  individually; at termination on day 21 post-infection, hearts and pancreata were collected for histology  of animals infected with the wt CVB3, and 80% (8/10) of animals infected with pBRCVB3 had myocardial lesions as indicated by inflammatory foci of 34.2 ± 13.4 and 11.8 ± 7.3, respectively (Table p ≤ 0.0001)  of mice infected with wt CVB3 had myocardial lesions 34.5 ± 13.3), whereas inflammatory changes were lacking in both vaccine-alone and vaccine/challenged groups , which involved assessment of percent neutralization of wt CVB3 in Vero cells exposed to serum collected from saline or Mt 10 vaccinated groups. Figure 18.To that end, we measured total Ig, IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, and IgE by enzyme-linked immunosorbent assay (ELISA) using CVB3 VP1 as a specific viral antigen, with Keyhole limpet hemocyanin (KLH) serving as a negative control, and we made a few observations Fig. b. (i) Se12. Using these tools, we analyzed virus-specific T cells in animals injected with saline or vaccinated with Mt 10 virus. In brief, a pool of spleens and lymph nodes, hereafter termed lymphocytes, harvested from these groups were stimulated with VP1 681–700 or VP1 721–740 peptides, and staining was performed by flow cytometry using IAk/VP1 681–700 dextramers and VP1 721–740 tetramers and their corresponding controls (Bovine ribonuclease [RNase] 43–56)12. As expected, CD4 T cells from saline-recipient animals did not bind VP1 681–700 or VP1 721–740 dextramers or tetramers, and their staining intensities were comparable to those of RNase 43–56 (control)  and VP1 721–740 tetramers , whereas staining for RNase 43–56 was negligible , monocyte chemoattractant protein (MCP)-1 (p ≤ 0.001), and keratinocyte-derived chemokine (KC) (p ≤ 0.05) on day 4 after vaccination, whereas granulocyte–macrophage colony-stimulating factor (GM-CSF) (p ≤ 0.001) was detected at day 21 when compared with saline recipients  1, Th2, and Th17, including interleukin (IL)-6, tumor necrosis factor (TNF)-α, and anti-inflammatory (IL-10) cytokines. The analyses revealed detection of mainly interferon (IFN)-γ and IL-2 [Th1], followed by IL-22, representative of Th17 subset, and IL-5, IL-4, and IL-13 (Th2) in cultures derived from vaccine-recipients as compared to saline-recipient animals without antigenic stimulations, suggesting that the virus-primed lymphocytes can spontaneously produce these cytokines  as a cardiac injury marker21. Using T-cell proliferation assay, we noted myosin reactivity in animals infected with the wt CVB3, as indicated by the appearance of T cells responding to cardiac myosin heavy chain (Myhc)-α 334–352 (p ≤ 0.001) , but not in the vaccine or vaccine/challenge groups. Taken together, the data suggest that the Mt 10 virus, despite being live attenuated, is unlikely to induce side effects described above, since cardiac injury is expected to occur as a consequence of direct virus-induced damage to the cardiac tissue24.We had previously reported that CVB3 infection can lead to the induction of pathogenic T cells reacting to cardiac myosin as a secondary event25. We sought to evaluate this phenomenon by looking for viral RNA in hearts and pancreata harvested from animals inoculated with wt CVB3, vaccine alone, and vaccine-challenged groups. This was achieved by quantitative/real-time RT-PCR using the probes and primers specific to CVB3-VP1. Expectedly, hearts and pancreata obtained from the wt CVB3 alone group contained CVB3 viral nucleic acids (p ≤ 0.01)  Fig. . Importa01) Fig. , suggest33. But, none of these approaches evaluated for their ability to prevent both myocarditis and pancreatitis33 except one modified live mutant virus (CVB3/H3)34, but in the latter, viral titers were still present in the hearts. Additionally, side effects such as autoimmunity were also not investigated35 since attenuated vaccine strains may retain a low degree of virulence, causing tissue damage in target organs such as heart, leading to autoimmunity as we have previously reported with the wt CVB319. Furthermore, to effectively prevent viral infections, induction of both antibody and T cell responses is critical because antibodies are needed to neutralize the viruses, and elimination of established infections requires participation of T cells. Therefore, ideally, vaccines are expected to induce both antigen-specific antibody and T cell responses and be free of side effects such as autoimmunity.In this report, we describe the creation and characterization of an avirulent strain of CVB3 as a live attenuated vaccine to prevent both myocarditis and pancreatitis induced by the wt CVB3 virus. Experimentally, various vaccine strategies have been tested with varied efficacies12. However, by localizing these epitopes within the viral canyon, we observed that VP1 771–790 had the potential to interact with the CAR, one of the receptors needed for viral entry into target cells36. Of note, virus entry requires attachment of CVB3 to CD55  which then facilitates interaction with the CAR38. Furthermore, interaction of CAR with CVB3, but not with CD55 alone leads to lytic infection39. Since, we wanted to preserve the virus entry to be able to induce immune responses by unleashing the critical viral proteins, but not lytic infection leading to tissue injury, we chose not to target and disrupt the virus attachment to CD55. As illustrated  and demonstrated that two of these (VP1 681–700 and VP1 721–740) could be used to analyze antigen-specific T cell responses in multiple CVB serotypesOur approach to create mutant viruses was unique in that we specifically targeted the buried amino acids of the viral capsid canyon, which are less likely to interact with the CAR, leading to the recovery of infectious viruses from five clones, Mt 2, Mt 3, Mt 4, Mt 8, and Mt 10  of the five mutant viruses failed to induce both myocarditis and pancreatitis, whereas others  induced mild myocarditis, if any; however, infected animals consistently developed pancreatitis, except that incidence and severity of pancreatitis induced with the Mt 8 virus were low , and IL-22 (Th17) were elevated in culture supernatants from vaccine recipients , as well as GM-CSF. Reports indicate that IP-10 enhances immune response against influenza infections51, and KC has been reported to be elevated for protection during Theiler’s murine encephalomyelitis virus infection52. Similarly, MCP-1 can promote broad-spectrum anti-viral effects in Dengue virus and Japanese encephalitis virus infections53, and GM-CSF can attenuate viral replication54 and also act as an adjuvant in therapeutic vaccines55. Thus, a combination of chemokines and T cell derived cytokines might contribute towards an anti-viral defense mechanism to Mt 10 virus. However, failure to detect classic anti-viral innate cytokines (IFN-α/β) might be because we analyzed these cytokines after day 4 post-vaccination. Their presence may become evident at earlier time points, which we have not examined in this study. Finally, by investigating the safety aspects of Mt 10 virus, we noted no generation of autoimmune response, which otherwise would be expected in wt CVB3 infection, as we have previously demonstrated19. Lack of increase in the cardiac injury marker cTnI in vaccine recipients lent further support to the proposition that the Mt 10 virus could be safely used as a vaccine virus.By investigating the vaccine characteristics of Mt 10 virus, we made three observations. First, priming animals with a single dose of the Mt 10 virus was sufficient to prevent infection by a lethal dose of wt CVB3 in challenge studies, and infected animals developed neither myocarditis nor pancreatitis Fig. . Second,56. As such, development of a vaccine for each serotype is challenging, and such an approach may not be viable practically. Thus, we intend to assess whether vaccine responses induced by the Mt 10 virus can also offer cross-protection against multiple serotypes of CVB; indeed, our preliminary data support this possibility (data not shown). Finally, reports indicate that CVB3 has been used to deliver foreign genes of vaccine importance or cytokines in vivo60. To this end, utility of the Mt 10 virus can be exploited that offer an added advantage in vaccine studies.In summary, we have described a novel attenuated strain of CVB3 called Mt 10 virus that serves as a vaccine candidate in the prevention of both myocarditis and pancreatitis in the mouse model of CVB3 infection. The Mt 10 virus induces IgG nABs accompanied with the generation of IFN-γ-producing antigen-specific T cells; vaccine recipients had no detectable viral RNA, indicating that the vaccine responses can effectively prevent infection in challenge studies. Translationally, there is merit in using the Mt 10 virus as a vaccine candidate to prevent CVB3 infection. But it should be noted that six CVB serotypes exist, and more than one serotype can induce similar diseasea) were procured from the Jackson Laboratory  and maintained according to the Institutional guidelines of the University of Nebraska-Lincoln, Lincoln, NE, and approval for animal studies was granted by the Institutional Animal Care and Use Committee, UNL . The study was carried out in compliance with the ARRIVE guidelines61. Infection studies were performed based on biosafety level 2 guidelines. When animals were found to have persistent clinical signs, such as failure to move when physically touched or prodded, or did not eat or drink, they were euthanized using a carbon dioxide chamber as recommended by the Panel on Euthanasia of the American Veterinary Medical Association.Six-to-eight-week-old male and female A/J mice . The purity of peptides was ascertained to be more than 90% via high-performance liquid chromatography, and their identity was confirmed by mass spectroscopy. Ultra-pure water or phosphate-buffered saline (PBS) was used to dissolve the peptides, and multiple aliquots of peptides were stored at  − 20 °C until further use. Full-length CVB3 VP1  and KLH protein  were procured commercially.16 , the footprints of binding region between virus and CAR were retrieved to localize the VPs 1 to 4. Potential interactions of amino acid residues within the VP1 771–790 with the host receptor (CAR) were analyzed using the BIOVIA Discovery Studio v 4.0 .By using the published crystal structure of CVB3 (PDB: 1COV)11. The buried amino acid residues within VP1 771–790 were targeted for site-directed mutagenesis. The nucleotides (nts) for amino acid residues at VP1 775, 776, 777, 780, 786, 787, 789 and 790 were replaced with the nts for alanine (A) residues, whereas the nts for A at VP1 788 were changed to G individually, yielding a total of nine mutant clones. Two additional mutants were derived by combining mutations together at residues 775 to 777 (Mt 5) for A, or residues 786 to 790 (Mt 11) for A or G as described above. The PCR products containing the mutations were cloned into the full-length infectious cDNA clone11, from which in vitro RNA transcripts were transcribed using T7 RNA polymerase at 37 °C, as recommended 11. Vero cells were grown to 80% confluency, then trypsinized and washed twice with 1 × PBS by centrifugation at 400xg for 6 min at room temperature (RT). After cells were resuspended in electroporation buffer  to a cell density of 10 × 106 cells/ml, the in vitro transcribed viral RNA (6 µg) was transferred to a 0.2 cm electroporation cuvette (BioRad), and 200 µl of cell suspension was added. The mixture was subjected to electroporation using Gene Pulser Xcell Electroporation System at 160 V, according to the manufacturer’s recommendations (BioRad). The electroporated cells were gently aspirated and transferred to 6-well plates containing 2 ml of fresh EMEM/10% fetal bovine serum (FBS) prewarmed to 37 °C. Medium was removed after 16 h, cells were washed with 1 × PBS, and replaced with 2 ml of fresh EMEM/2% FBS, then incubated up to 5 days. As the CPE became evident, supernatants containing mutant virus were harvested, passaged, titrated, and stored in aliquots at -80 °C till further use.CVB3 mutant viruses were generated by site-directed mutagenesis using a CVB3-infectious clone, pBRCVB3, developed previously in our laboratory62. Briefly, Vero cells (0.25 × 106 cells/ml/well) were plated on to sterile coverslips in 12-well plates and incubated at 37 °C. After adhesion of Vero cells, the monolayers were washed with 1 × PBS and infected with wt CVB3 , pBRCVB311 (positive controls), and mutant viruses at an MOI of 0.5 for 1.5 h. After adsorption, inoculum was aspirated and replaced with EMEM supplemented with 2% FBS (ATCC). Cells grown in medium alone were used as negative controls. Cells were incubated for 12 h at 37 °C, then washed and fixed in methanol/acetone (1:1) for 40 min at RT. They were then washed three times with 1 × PBS and incubated with anti-CVB3 serum (1:200) in PBS containing 2.5% bovine serum albumin for one hour at RT. After five washings with PBS/Tween 20 , cells were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary rabbit anti-horse IgG  for one hour at RT. Finally, coverslips containing the cells were washed, mounted, and visualized under a Nikon A1-Eclipse 90i confocal microscope system . Images were acquired sequentially at an excitation/emission wavelength of 561.5 nm/553–618 nm laser set up for pseudo-colored green channel at 60 × magnification.We used CVB3 anti-serum  to verify the infectivity of viruses derived from mutant clones, allowing us to detect cells expressing viral proteins based on immunofluorescence11. After the CPE was confirmed, culture supernatants containing virus were harvested, and viral stocks were stored at  − 80 °C in aliquots until further use. The viruses were titrated, and tissue culture infective dose 50 (TCID)50 values were determined according to the Spearman-Karber method63. To compare the growth characteristics of the wt CVB3, pBRCVB3, and mutant viruses, Vero cells grown in 12-well plates were infected in triplicates at MOIs 0.1 and 3.0. Culture supernatants were collected at various days post-infection and virus titers were determined as above.Vero cells (ATCC) grown to 80–90% confluency were infected with wt CVB3, pBRCVB3, and mutant viruses as described previously50/200 µl, and the inocula were administered into A/J mice intraperitoneally (i.p). The control (uninfected) mice received only 1 × PBS. Mice were housed 2 to 3 per cage in filter-top cages assembled with closed air-circulation. Cages containing the chow diet and waterers were changed every 3 days until the end of the experiment. The animals had ad libitum access to food and water during the entire study period. Animals were inspected twice daily, and body weights were recorded every 1 to 2 days. An alternative food and fluid source, trans gel diet , was placed on the cage floor as needed. Hearts and pancreata were collected from animals that died naturally. At termination (day 21 post-infection), animals were euthanized, and hearts, and pancreata were collected.For infection studies, the virus stock was diluted in 1 × PBS to contain 10,000 TCID64. The sections were blinded to treatment and examined by board-certified pathologist, Dr. David Steffen. Pathology scores were generated by enumerating the inflammatory foci, necrosis, mineralization, and fibrosis. Individual scores were used to compare the qualitative nature of the lesions. Total scores represented total foci of pathologic change across the three sections of heart. Multiple changes present in a single focus were counted as 1 in the total count65. The severity of pancreatic change was estimated as percent of tissue section involvement from one random section of pancreas. The nature of pancreatic lesions was noted as atrophy, inflammation, mineralization, and necrosis or a combination of these65.Hearts and pancreata were fixed by immersion in 10% phosphate-buffered formalin. Each tissue was sliced into three representative 5 µm cross-sections and stained with hematoxylin and eosin (H and E)6 TCID50/200 µl in 1 × PBS, i.p.) on day 0. After 14 days, animals were injected with saline or wt CVB3 , and body weights were taken every two days thereafter. Hearts and pancreata were collected from animals that died naturally. At termination , animals were euthanized, and serum, spleens, lymph nodes, hearts, and pancreata were collected for further analysis.Groups of mice were administered Mt 10 virus . An equal volume of wt CVB3 suspension containing 100 TCID50/ml was incubated with serially diluted sera at 37 °C for 1 h in a humidified chamber with 5% CO2. After incubation, 100µl of the mixture of each dilution was added in quadruplets to plates containing monolayers of cells and incubated at 37 °C. After four days, plates were observed for CPE, and the highest serum dilution that showed protection from CPE was considered to be the neutralization titer.A virus neutralization test was performed using the sera obtained from different groups. Vero cells were plated at 0.25 × 1067. In brief, 96-well polystyrene microtiter plates were coated with or without CVB3 VP1 or an irrelevant control (KLH) (5 μg/ml) in 1 × coating buffer  and incubated at 4 °C overnight. Plates were washed with 1 × PBS/0.05% Tween-20 and blocked with 1 × PBS/2% BSA/5% normal goat serum for 1.5 h at RT (1:150), and then serum samples were added in duplicates. The plates were incubated at 37 °C for 1 h, and then washed. Horse-radish peroxide (HRP)-labeled goat anti-mouse IgA, IgE, IgM, IgG1, IgG2a, IgG2b, IgG3, and total Ig were then added as secondary antibodies . After the plates were incubated at RT for 2 h, 1 × tetramethylbenzidine (TMB) solution was added as a substrate (eBioscience) and reactions were stopped using 1 M phosphoric acid. Plates were read at 450 nm using an automated ELISA reader , and optical density (OD) values were measured68.Serum samples collected from groups of mice that received Mt 10 virus or saline on days 14 and 35 were analyzed for total Ig, IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, and IgE as described previouslyk tetramers and dextramers to enumerate the frequencies of CD4 T cells specific to VP1 681–700 and VP1 721–74012. In this study, we used IAk/dextramers for VP1 681–700 and IAk/tetramers for VP1 721–740, which were available at the time. Briefly, single cell suspensions of lymphocytes were obtained from mice injected with Mt 10 virus or saline. Cells were stimulated with VP1 681–700 and VP1 721–740 (20 µg/ml) for 2 days, and viable cells were maintained in medium containing IL-212. During the 7 to 10 days post-stimulation, cells were stained with the VP1 dextramers and tetramers described above, as well as their corresponding control dextramers and tetramers (RNase 43–56), followed by anti-CD4  and 7-aminoactinomycin D 71. After acquiring the cells by flow cytometry , percentages of dextramer or tetramer positive cells were determined in the live (7-AAD¯) CD4+ subset using FlowJo software [v 7.6.5] 12.We recently created MHC class II/IASupernatants obtained from lymphocyte cultures prepared from animals injected with saline or Mt 10 virus were stimulated with or without VP1 681–700 and VP1 721–740 (20 μg/ml) on day 3 post-stimulation. Cytokine analysis was performed using the LEGENDplex Murine Th cytokine Panel . Cytokine analysis included a panel of IL-2, IFN-γ, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, and TNF-α. Similarly, serum samples obtained from saline and Mt 10 virus groups were evaluated for a panel of anti-viral cytokines and chemokines using LEGENDplex Murine anti-viral kit . These included IFN-γ, KC, TNF-α, MCP-1, IL-12p70, RANTES, IL-1β, IP-10, GM-CSF, IL-10, IFN-β, IFN-α, and IL-6. Standard curves were obtained by serially diluting the lyophilized mouse cytokine standard mix provided in the kit. Briefly, capture bead/cytokine antibody conjugates were first prepared, and the mixtures were added to a tube containing diluted standards or test samples, followed by addition of detection antibodies and streptavidin–phycoerythrin reagents. After acquisition by flow cytometry, cytokine concentrations were determined using the LEGENDplex data analysis software suite (BioLegend).3[H] thymidine-incorporation assay73. In brief, cells were suspended in RPMI containing 10% FBS, 1 mM sodium pyruvate, 4 mM l-glutamine, 1 × each of non-essential amino acids and vitamin mixture, and 100 U/ml penicillin–streptomycin . Cells were stimulated with Myhc-α 334–352 (50 µg/ml) or RNase 43–56 (50 µg/ml) for 2 days at a density of 5 × 106 cells/ml in triplicates, whereas cells cultured with no peptides were used as medium controls. After pulsing with 3[H] thymidine  for 16 h, proliferative responses were measured as counts per minute (cpm) using a Wallac liquid scintillation counter .Lymphocytes obtained from groups of animals that received wt CVB3 or Mt 10 virus and Mt 10 virus/wt CVB3 were used to assess Myhc-α 334–352-reactive T cell responses based on tritiated 10 of their concentration.Serum cTnI levels were measured in the sera collected from saline, wt CVB3, Mt 10 virus, and Mt 10 virus/wt CVB3 groups using the high-sensitivity cTnI ELISA kit according to manufacturer’s recommendations . Briefly, 100 μl of cTnI HRP conjugate and 100 μl of standards/samples were added to anti-cTnI-coated wells. After mixing, plates were incubated on an orbital shaker for 1 h at RT, followed by addition of 100µL TMB as substrate and an equal volume of stop solution. Plates were read at 450 nm using an automated ELISA reader to obtain the OD values (BioTek instruments). Concentration of cTnI in unknown samples was calculated using the standard curve generated by plotting the absorbance values of the standards versus logCt)−. RNA was isolated using the RNeasy kit , and samples were treated with deoxyribonuclease (DNase) I and quantified using the NanoDrop ND-1000 spectrophotometer . In a single-step reaction, RNA was reverse-transcribed and PCR was performed using the iTaq Universal one-step RT-qPCR kit . The real-time quantitative PCR analysis included amplifications for CVB3 VP1 (target gene) and glyceraldehyde-3-phosphate dehydrogenase  using TaqMan Gene Expression Assays (Applied Biosystems) and the CFX96 Touch Real-time PCR detection system (BioRad). Expression of CVB3 VP1 was normalized to GAPDH using the 2+ and dextramer+ cells, antibodies, cytokines, T cell proliferation, cTnI, and qPCR were analyzed by unpaired student’s t-test, Mann–Whitney test, or Kruskal–Wallis test for pairwise comparisons between the groups. Two-way ANOVA with Sidak’s post-test was used to compare the body weight changes. Log-rank test with Bonferroni correction was used to analyze statistical significance of the survival curves. Barnard’s exact test was used to analyze the histological parameters74. Graphs were prepared by GraphPad Prism software v8.0.Statistical analyses were performed using GraphPad Prism software v8.0 . Data sets pertaining to viral titers, tetramerSupplementary Information.


Per2) gene expression is essential for circadian oscillation, nutrient metabolism, and intestinal microbiota balance. Herein, we combined environmental LD cycles with Per2 gene knockout to investigate how LD cycles mediate Per2 expression to regulate colonic and cecal inflammatory and barrier functions, microbiome, and short-chain fatty acids (SCFAs) in the circulation. Mice were divided into knockout (KO) and wild type (CON) under normal light–dark cycle (NLD) and short-light (SL) cycle for 2 weeks after 4 weeks of adaptation. The concentrations of SCFAs in the serum and large intestine, the colonic and cecal epithelial circadian rhythm, SCFAs transporter, inflammatory and barrier-related genes, and Illumina 16S rRNA sequencing were measured after euthanasia during 10:00–12:00. KO decreased the feeding frequency at 0:00–2:00 but increased at 12:00–14:00 both under NLD and SL. KO upregulated the expression of Per1 and Rev-erbα in the colon and cecum, while it downregulated Clock and Bmal1. In terms of inflammatory and barrier functions, KO increased the expression of Tnf-α, Tlr2, and Nf-κb p65 in the colon and cecum, while it decreased Claudin and Occludin-1. KO decreased the concentrations of total SCFAs and acetate in the colon and cecum, but it increased butyrate, while it had no impact on SCFAs in the serum. KO increased the SCFAs transporter because of the upregulation of Nhe1, Nhe3, and Mct4. Sequencing data revealed that KO improved bacteria α-diversity and increased Lachnospiraceae and Ruminococcaceae abundance, while it downregulated Erysipelatoclostridium, Prevotellaceae UCG_001, Olsenella, and Christensenellaceae R-7 under NLD in KO mice. Most of the differential bacterial genus were enriched in amino acid and carbohydrate metabolism pathways. Overall, Per2 knockout altered circadian oscillation in the large intestine, KO improved intestinal microbiota diversity, the increase in Clostridiales abundance led to the reduction in SCFAs in the circulation, concentrations of total SCFAs and acetate decreased, while butyrate increased and SCFAs transport was enhanced. These alterations may potentially lead to inflammation of the large intestine. Short-light treatment had minor impact on intestinal microbiome and metabolism.Regular environmental light–dark (LD) cycle-regulated period circadian clock 2 ( Per2) gene is wildly distributed in the central nervous system such as the brain and spinal cord, or surrounding organs, such as the skin and gastrointestinal tract  axis  cycles . As the al tract . MutatioLs) axis .PER2 in reindeer caused a loss of binding ability with cryptochrome 1 (CRY1), resulting in arrhythmicity, thus adapting the reindeer to the drastic LD changes in polar regions propionic acid (4-OH-PPA) and 3-phenylpropionic acid (PPA)  . Gut micA)  gene is lost when the rhythm phase is advanced by 6 h in mice (Per1) expression late at night and strong Cry1 expression at midnight in wild-type mice under SL. In addition, there was a high Cry1 expression in Per2 mutant mice at midnight under SL, which suggested that the LD cycle of 8L:16D also causes rhythm disorders. Besides, SL cycle is more related to the reproduction of animals , thus regulating circadian oscillation and impacting the gut microbiome and nutrient metabolism. Therefore, Per2 knockout (KO) and wild-type (CON) mice were managed under NLD and SL, respectively, for 2 weeks after adaption for 4 weeks, in order to investigate how Per2 regulates colonic and cecal inflammatory functions, barrier functions, gut microbiome, and SCFAs circulation in response to different LD cycles.Herein, we hypothesized that All animal experiments were performed according to the ethical policies and procedures approved by the Animal Care and Use Committee of Yangzhou University, Jiangsu, China  2017-0044).Per2 gene knockout (+/−Period2) based on CRISPR/Case9 technology were provided by Biocytogen Biotechnology Co., Ltd. . Mice were then sent to the Institute of Neuroscience, Chinese Academy of Science, non-human primate research platform , to expand the scale of reproduction, in order to obtain a sufficient number of positive homozygous mice  and wild-type mice . Each 8-week-old mouse was in good health, similar in body size and initial body weight. KO and CON mice were then randomly divided into alternating photoperiods of normal light–dark cycle treatment  and short-light cycle treatment  (The heterozygous C57BL/6N mice with systemic arkness) , 33, 34;Mice were fasted 1 day before sampling; then, blood was collected from the retroorbital sinus for measurements of concentrations of SCFAs. Mice were then anesthetized with ether and euthanized by spinal dislocation during 10:00–12:00 a.m. The colon and cecum tissues were harvested, rinsed in phosphate buffer solution (PBS), and rapidly frozen in liquid nitrogen for measurements of the expressions of circadian rhythm, SCFAs transporter, and inflammatory- and barrier-related genes following a previously described protocol . Colonic2O to make the volume of 20 µl. Reaction procedure was set as 42°C for 15 min and 95°C for 3 min following the manufacturer’s instructions.Total RNA was extracted from colonic and cecal epithelial tissues using the FastPure Cell/Tissue Total RNA Isolation Kit V2 . Concentration and purity of total RNA were determined with a NanoDrop spectrophotometer . Reverse transcription referred to FastKing gDNA Dispelling RT Super Mix . Reverse transcription reaction system was as follows: 5× FastKing-RT Super Mix, 4 µl; total RNA, 1,000 ng; and RNase-free ddH2O to make the volume of 20 µl. PCR reaction procedure was set as 95°C for 60 s, 40 cycles of 95°C for 10 s and 60°C for 30 s, and, ultimately, the test was set as 95°C for 15 s, 60°C for 60 s, 95°C for 30 s, and 60°C for 15 s. The standard curve method and QuantStudio™ 7 Flex Real-Time PCR Software  were used for data analysis. Relative expression of the target gene was calculated using 2−ΔΔCt methods  using the 2× TSINGKE Master qPCR Mix (SYBR Green I)  in an ABI7500 (Thermo Fisher) sequence detector. The reaction system for PCR was as follows: qPCR Mix, 10 µl; forward primer, 0.8 µl (10 µM); reverse primer, 0.8 µl (10 µM); 50× ROX Reference Dye, 0.4 µl; and ddH methods . SpecifiTotal protein was extracted from colonic and cecal epithelial tissues using radioimmunoprecipitation assay (RIPA) lysis buffer , which contained 1 mM dilution of phenylmethylsulfonyl fluoride . Total protein was extracted according to the manufacturer’s instructions, and the concentration of protein was determined with the Enhanced BCA Protein Assay Kit . Protein was denatured at 95°C for 10 min prior to Western blotting.Western blot method was used to determine the protein expression of Per2. Details of Western blot were reported previously by our laboratory . Brieflyg for 10 min. The supernatant was collected and filtered through a 0.25-μM syringe filter. A total of 0.4 ml metaphosphoric acid  was added, and then, the entire contents were vortexed, centrifuged, and filtered, and the supernatant was collected for subsequent analyses. A standard mix (0.4 μl) contains 55.06 mmol/L of acetate, 15.84 mmol/L of propionate, 4.25 mmol/L of isobutyrate, 8.57 mmol/L of butyrate, 3.58 mmol/L of isovalerate, and 3.56 mmol/L of valerate, and test samples were mixed and ran through a CP-WAX capillary column  in a gas chromatograph . The SCFAs were determined according to the preliminary method of our laboratory  was used to extract total microbial DNA. The concentration and purity of total DNA were determined by the NanoDrop spectrophotometer . Illumina Hiseq2500-PE250 sequencing platform was used to determine the abundance and diversity of the intestinal content microbiota. The method amplified the bacterial 16S ribosomal RNA (rRNA) region using the following primers: primer  target V3–V4 hypervariable region was used for sequencing . Sequencvia Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Paired-end reads generated from Illumina platforms were processed and merged using FLASH software (v1.2.7) . Then, hdatabase .p < 0.05, significant difference; ** or ***denotes p < 0.01 or p < 0.001, extremely significant difference). Bacterial taxa analyses were plotted using the R package “ggplot2” based on the modified OTUs data. The Venn diagrams were analyzed using the R package “VennDiagram.” PCoA diagram was carried out by R package “vegan.” The bubble diagram of pathway enrichment analysis was made using the R package “ggplot2.” The bacterial cluster results were carried out by R package “pheatmap.” The R version was 4.0.2, and R packages were described previously  software. GraphPad Prism 6.0 software was used to draw histograms. Results are presented as means ± standard error of the mean (SEM)  but increased at 12:00–14:00 (p < 0.05 or p < 0.001) both under NLD and SL (p > 0.05). Short-light treatment also did not alter the daily feeding rhythm compared with NLD of KO and CON mice (p > 0.05). The feeding rhythm of CON mice showed trends that were higher at night but lower during the day, while KO mice showed the opposite.Data indicated that the feeding frequency of Per2 knockout did not have a significant impact on the concentrations of total SCFAs, acetate, propionate, butyrate, and acetate/propionate (p > 0.05) compared with CON mice (p > 0.05). There was no interaction effect on the serum SCFAs indicators (p > 0.05).Data indicated that Per2 knockout significantly reduced the concentrations of total SCFAs (p < 0.001), acetate (p = 0.001), propionate (p = 0.011), and increased butyrate (p = 0.021), but did not impact isobutyrate, isovalerate, valerate, and acetate/propionate (p > 0.05) in colonic contents (p > 0.05), except reducing isobutyrate (p < 0.001) and isovalerate (p = 0.031) compared with NLD. The interaction between two treatments did not impact the concentration of SCFAs (p > 0.05). In terms of the concentration of SCFAs in cecal content, KO reduced total SCFAs (p = 0.020) and acetate (p < 0.01) but increased butyrate (p = 0.001); no differences were observed in propionate, isobutyrate, isovalerate, valerate, and acetate/propionate (p > 0.05). Short light did not impact SCFAs indicators (p > 0.05) compared with NLD in cecal content. Besides, the interaction between two treatments significantly impacted the concentration of propionate (p < 0.01).Compared with CON mice, Per2 knockout mice both under NLD and SL in colonic epithelium (p < 0.05 or p < 0.001). There was a high knockout efficiency of Per2 gene in colon and cecum tissues. In terms of expression of other circadian rhythm genes during 10:00 to 12:00 a.m., KO upregulated expression of Per1 and Rev-erbα gene but downregulated circadian locomotor output cycles kaput (Clock) and Bmal1 both under NLD and SL. The KO also increased the expression of period circadian clock 3 (Per3) gene under NLD in colonic epithelium  compared with CON mice. For cecal epithelium, KO upregulated expression of Per1, cryptochrome 2 (Cry2), and Rev-erbα but downregulated expression of Clock and Bmal1 genes both under NLD and SL. The KO also downregulated expression of Cry1 gene under SL  compared with CON mice. Short-light treatment did not impact expression of colonic and cecal epithelial circadian rhythm genes (p > 0.05) compared with NLD.Compared with CON mice, mRNA and protein expression of Per2 significantly decreased in Per2 knockout significantly upregulated expression of Na+/H+ exchanger 1 (Nhe1), Na+/H+ exchanger 3 (Nhe3), monocarboxylic acid transporters 1 (Mct1), and monocarboxylic acid transporters 4 (Mct4)  but did not impact expression of Na+/H+ exchanger 2 (Nhe2), anion exchanger 2 (Ae2), and +/K+ ATPaseNa (p > 0.05) both under NLD and SL in colonic epithelium (Nhe3 gene compared with NLD in KO mice (p < 0.01)  but did not impact expression of Nhe2, Mct1, Ae2, and +/K+ ATPaseNa (p > 0.05) both under NLD and SL in cecal epithelium (p > 0.05).Compared with CON mice, Per2 knockout significantly upregulated expression of tumor necrosis factor α (Tnf-α) gene, while it downregulated expression of tight junction protein 1 (Zo-1), Claudin-1, and Occludin genes (p < 0.05 or p < 0.01) but did not impact the expression of interleukin 1β (Il-1β) and interleukin 6 (Il-6) genes (p > 0.05) both under NLD and SL in colonic epithelium . However, it did not impact expression of Il-6 and Zo-1 genes (p > 0.05) both under NLD and SL (p > 0.05).Compared with CON mice, Per2 knockout significantly upregulated expression of toll-like receptor 2 (Tlr2) and nuclear factor κb (Nf-κb) p65 genes  but did not impact expression of toll-like receptor 4 (Tlr4) and Myd88 genes (P > 0.05) both under NLD and SL in colonic epithelium  but did not impact expression of Myd88 (p > 0.05) both under NLD and SL (p > 0.05), except upregulating the expression of Tlr2 gene of KO mice in cecal epithelium (p < 0.001).Compared with CON mice, Per2 knockout significantly altered intestinal bacterial β-diversity, while short-light treatment did not have an impact. Lastly, the α-diversity (p<0.05) both under NLD and SL of colonic and cecal microbiome, except that KO had no impact on the Simpson index of cecal content (p > 0.05). Short light did not impact bacterial α-diversity (p > 0.05).Lachnospiraceae NK4A136, Eubacterium_xylanophilum, Roseburia, Lachnospiraceae UCG_006, Ruminiclostridium, Anaerotruncus, Rikenella, Candidatus Saccharimonas, and Ruminococcaceae UCG_003 genus were enriched in NLD+KO; Erysipelatoclostridium, Prevotellaceae UCG_001, Olsenella, and Christensenellaceae R_7 genus were enriched in NLD+CON; Escherichia Shigella, Lactobacillus, Eubacterium coprostanoligenes, Turicibacter, Prevotellaceae NK3B31, and Marvinbryantia genus were enriched in SL+KO; and Parasutterella, Allobaculum, Bacteroidales S24-7 unidentified, and Enterorhabdus genus were enriched in SL+CON. The cluster results  of KO mice were significantly higher than those of CON mice both under NLD and SL. However, Thaiss et al. ”, etc. were also enriched, which indicated that most bacteria were involved in amino acid, carbohydrate, and energy metabolism. Several previous studies showed that Per2 interacts with key nuclear receptors    and playev-erbα) . Per2 mud levels , 60. Perfat diet , 51. Thi pathway , 62.Per2 knockout altered the metabolism of propionate and butyrate. For further verification, the proportion of SCFAs in the colon and cecum were measured. KO hadno impacts on SCFAs in the serum; the probable reason may be that SCFAs were more energy substance for the intestinal epithelial cells rather than transported into the blood, such as butyrate. KO decreased the concentrations of total SCFAs and acetate in the colon and cecum but increased butyrate. In other words, the increased abundance of Lachnospiraceae and Ruminococcaceae in KO was the main reason for the increased concentrations of butyrate. Previous reports also indicated that the above bacteria were closely related to the synthesis of SCFAs. For example, Lachnospiraceae NK4A136 and Rosebuira genus participated in the production and metabolism of butyrate and maintenance of intestinal barrier functions  resulted in the upregulation of MCT1 abundance, which was consistent with this study. Meanwhile, the abundance of PER2 mRNA and protein increased when 15 mM sodium butyrate was added to stimulate goat GRECs in vitro, indicating that absorption of SCFAs regulated by PER2 may be mediated by butyrate  after Per1/2 gene double knockout. Not only that, we need to monitor the dynamic alterations in microbial and metabolic rhythms during a 48 h period.In short, our data are consistent with the hypothesis that Per2 knockout dysregulated the TTFLs axis and altered the expression of circadian genes such as upregulating Per1 and Rev-erbα and down-regulating Bmal1 and Clock in colon and cecum tissues of mice. Circadian rhythm disorder further induced the loss of feeding rhythm, which in turn led to alterations in gut microbiome. KO increased the α-diversity of gut microbiome, and the abundance of Lachnospiraceae and Ruminococcaceae were significantly upregulated, which further led to altering SCFAs metabolism and transport. Data indicated that KO also increased the concentrations of butyrate but decreased total SCFAs and acetate; the transport was enhanced because of the upregulation of Nhe1, Nhe3, Mct1, and Mct4 genes. This may potentially lead to inflammation of the large intestine. However, short-light treatment had little impact on intestinal microbiome and metabolism according to our data.In conclusion, based on the present work, The 16S rRNA gene amplicon sequencing data generated during the current study were submitted to NCBI under BioProject PRJNA750583.All animal experiments were performed according to the ethical policies and procedures approved by the Animal Care and Use Committee of Yangzhou University, Jiangsu, China  2017-0044).MW, LH, and YZ designed research; QX, LH, WW, JH, and YZ conducted research; YZ and LG, analysed data; YZ and LG wrote paper; MW, JJL, QY, and PZ reviewed and modified paper. MW and PZ had primary responsibility for final content. All authors read and approved the final manuscript.This study was supported by grants from the Natural Science Foundation of China (31672446); the funds from State Key Laboratory of Sheep Genetic Improvement and Healthy Production , Shihezi, China; and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.