Electroacupuncture improves low-grade duodenal inflammation in FD rats by reshaping intestinal flora through the NF-κB p65/NLRP3 pyroptosis pathway.

Electroacupuncture (EA) is an effective alternative for the treatment of functional dyspepsia (FD). It reduces low-grade duodenal inflammation and improves the symptoms of FD by downregulating the expression of NF-κB p65 and NLRP3, but its mechanism needs to be elucidated. To examine the regulatory effect of electroacupuncture (EA) on intestinal flora and NF-κB p65/NLRP3 pyroptosis pathway in FD rats. The FD rat model was established via multi-factor stress intervention for two weeks. The rats were randomly divided into the NC group, model group, NF-kB inhibitor group (NF-κB inhibitor BAY 11-7082 was administered), EA group, and EA + NF-kB inhibitor group. After 14 days of treatment, the rats were sacrificed, and the protein and mRNA levels of NF-κB p65, IκB, and NLRP3 in the duodenum were evaluated by Western blotting assays and real-time fluorescent quantitative PCR. The Illumina MiSeq sequencing platform was used to analyze the V4 region of the 16S rRNA gene of intestinal flora and predict functional genes. The concentration of short-chain fatty acids (SCFAs) in feces was assessed by metabolomics. EA can decrease low-grade duodenal inflammation and promote gastrointestinal motility in FD rats. This effect is mediated by inhibition of the NF-κB p65/NLRP3 pyroptosis pathway, an increase in the alpha and beta diversity of gut microbiota in the duodenum, an increase in the abundance of beneficial bacteria at the phylum and genus levels, and an increase in the content of SCFAs. The protective effect of EA against FD might involve multiple hierarchy and pathways. EA may remodel intestinal flora by inhibiting the NF-κB p65/NLRP3 pyroptosis pathway, thereby improving low-grade duodenal inflammation in FD rats.

Pellino1 orchestrates gut-kidney axis to perpetuate septic acute kidney injury through activation of STING pathway and NLRP3 inflammasome.

AIMS: Intestinal barrier dysfunction is the initial and propagable factor of sepsis in which acute kidney injury (AKI) has been considered as a common life-threatening complication. Our recent study identifies the regulatory role of Pellino1 in tubular death under inflammatory conditions in vitro. The objective of our current study is to explore the impact of Pellino1 on gut-kidney axis during septic AKI and uncover the molecular mechanism (s) underlying this process. MATERIALS AND METHODS: Immunohistochemistry (IHC) was conducted to evaluate Pellino1 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) levels in renal biopsies from critically ill patients with a clinical diagnosis of sepsis. Functional and mechanistic studies were characterized in septic models of the Peli-knockout (Peli1-/-) mice by histopathological staining, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence, biochemical detection, CRISPR/Cas9-mediated gene editing and intestinal organoid. KEY FINDINGS: Pellino1, together with NLRP3, are highly expressed in renal biopsies from critically ill patients diagnosed with sepsis and kidney tissues of septic mice. The Peli1-/- mice with sepsis become less prone to develop AKI and have markedly compromised NLRP3 activation in kidney. Loss of Peli1 endows septic mice refractory to intestinal inflammation, barrier permeability and enterocyte apoptosis that requires stimulator of interferons genes (STING) pathway. Administration of STING agonist DMXAA deteriorates AKI and mortality of septic Peli1-/- mice in the presence of kidney-specific NLRP3 reconstitution. SIGNIFICANCE: Our studies suggest that Pellino1 has a principal role in orchestrating gut homeostasis towards renal pathophysiology, thus providing a potential therapeutic target for septic AKI.

Electroacupuncture regulates hepatic cholesterol synthesis by HMGCR deubiquitination in rats.

This experiment was designed to explore the effect and mechanism of electroacupuncture (EA) for hyperlipidemia and hepatic cholesterol synthesis in rats. Liver and adipose tissues were assessed histologically, and body and liver weight, serum and liver lipid levels, expression of mTOR/ubiquitin-specific peptidase 20 (USP20)/recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and phosphorylation of mTOR and USP20 were measured. In vitro deubiquitination assays with liver cytosol were conducted. EA at Fenglong point ameliorated hyperlipidemia and hepatocyte steatosis, and decreased p-USP20, p-mTOR and HMGCR expression in the liver by reducing deubiquitination. Furthermore, EA decreased feeding-induced lipid biosynthesis in the liver. Concomitantly, EA prevented the induction of phosphorylated USP20 and mTOR, and HMGCR expression; and reduced the deubiquitination of HMGCR after re-feeding. This experiment demonstrated that EA can effectively improve hyperlipidemia and reduce hepatic cholesterol synthesis by counteracting the deubiquitination activity of HMGCR in hyperlipidemic rats.

[Effects of the cutaneous and blood contents of glucose on wound healing in diabetic rats with superficial partial thickness scalding].

OBJECTIVE: To study the effect of the cutaneous and blood contents of glucose on wound healing in diabetic rats with superficial partial thickness scalding. METHODS: 96 Sprague-Dawley rats were randomized into control group and STZ-induced diabetic group, inflicted with superficial partial thickness scalding of 10% TBSA on the back. The glucose content in the blood and wound tissue were evaluated before injury and at day 1, day 3, day 5, day 7, day 10 and day 14 after injury. Wound healing process in the rats was dynamically observed by naked eyes and histologically examined. The cell cycles of keratinocytes from wound site were determined before injury and at day 3, day 7 and day 10 after injury. RESULTS: The concentrations of blood glucose in diabetic wound was significant increased than normal group (27.28 mmol/L +/- 0.80 mmol/L vs. 4.65 mmol/L +/- 0.14 mmol/L, P < 0.01). The content of local glucose in wound tissue were significantly correlated with that in the blood (r = 0.881, P < 0.05). When compared with the control group, wound healing of the diabetic rats were delayed with the characteristics of impaired epithelialization and decreased. percentages of S stage and G(2)/M stages of keratinocytes. CONCLUSION: Local glucose content in diabetic wound was varied with blood glucose concentration. The increased glucose concentration in diabetic wound was attributed to the impaired wound healing in diabetes. High glucose concentration could inhibit the epithelialization and decrease the keratinocyte proliferation.

[Effects of advanced glycosylation end products on the biological behavior of neutrophils].

OBJECTIVE: To investigate the effects of advanced glycation end products (AGE) on the biological behavior of neutrophils in vitro, to look for the relationship between accumulation of AGE and abnormal inflammation in wound healing in diabetic mellitus patients. METHODS: Neutrophils were isolated from SD rats and incubated in vitro. The cells were divided into four groups according to different concentrations of AGE in cell suspension: control group (C, with treatment of RPMI - 1640), A group (with treatment of 0.315 mg/mL AGE + RPMI - 1640), B group (with treatment of 0.625 mg/mL AGE + RPMI - 1640), D group (with treatment of 1.250 mg/mL AGE + RPMI - 1640). Activity of neutrophils were determined by MTT colorimetric assay. Selectin-L mRNA expressions were analyzed by reversible transcription polymerase chain reaction (RT -PCR) technique. The levels of reactive oxygen species (ROS) in neutrophils were measured with DCFH-DA method. The protein concentration of neutrophil elastase (NE) was assayed by ELISA. RESULTS: The activity of neutrophils were obviously increased in A, B, and D groups when compared with that in C group [(0.170 +/- 0.040) in C group, (0.320 +/- 0.030) in A group, (0.380 +/- 0.020) in B group, (0.290 +/- 0.010) in D group, P <0. 05]. The expression of Selectin-L mRNA in A, B, D groups were significantly higher than that in C group (0.95 +/- 0.08, 1.36 +/- 0.27, 0.50 +/- 0.26.vs.0.36 +/- 0.26, P < 0.05. respectively). The ROS levels in A, B, D groups was markedly higher than that in C group (1.64 +/- 0.20, 2.16 +/- 0.26, 3.26 +/- 0.75. vs. 0.72 +/- 0.15, P <0.05, respectively). The levels of NE in A, B, D groups were significantly increased when compared with that in C group(1.98 +/- 0.43, 2.50 +/- 0.43, 2.01 +/- 0.18 vs 0.91 +/- 0. 21, P <0.05, respectively). CONCLUSION: AGE can enhance the activity of neutrophil, with change in cellular biological behaviors, which may be one of main reasons for abnormal inflammation in wounds of diabetes mellitus patients.

[Study of neovascularization disturbance in deep partial-thickness scald in rats with diabetes mellitus].

OBJECTIVE: To study the relationship between the degree of neovascularization and non-healing wounds in scalded rats with diabetic mellitus. METHODS: Sixty Sprague-Dawley rats were randomly divided into control group (C, n = 30, with treatment of isotonic saline) and streptozocin (STZ)-induced diabetic group (D, n = 30, with treatment of STZ), and then they were inflicted with 20% TBSA deep partial thickness scald. Wound specimens were harvested immediately after scald and on 1, 3, 7, 10, 14, 21 post scald days (PSD) to observe histological changes, and wound healing rates were calculated. Degree of neovascularization in wound (labeled with blue microsphere) and the quantity of vascular endothelial cells (labeled with red CD31) were also measured by double-labeling immunofluorescence. RESULTS: Compared with those in C group, Wound healing rate and histological value scores were lowered, and the degree of neovascularization was abated markedly at each time point. The degree of neovascularization in D group (12.00 +/- 1.40) was obviously lower than that in C group on 7 PSD (60.00 +/- 3.00, P <0.01). There was no obvious difference in the number of vascular endothelial cells in both groups, however, the majority of endothelial cells had not formed functional capillaries in D group. CONCLUSION: Vascular endothelial cell can proliferate actively with poor blood supply in diabetic nonhealing with deep partial-thickness scald wounds, but it is still poor in blood supply due to lack of functional capillaries.

[Study on the mechanism of scar formation: epidermis template defect theory].

Dermal defection and the degree of its loss determine the natural process of wound healing, which is the key reason leading to excess scar hyperplasia. The function of tri-dimensional structure in dermis acts as a template to regulate the properties of reparative cells. The template structure induces the reparative cells to grow into the structure which changes the skin mechanic status on wound area. Also, the component of extracellular matrix can affect behaviours of fibroblasts negatively or positively, for the reason that the structure of dermal tissue has a permissive effect on the dermal components in regulating behaviours of reparative cells. Therefore, the behaviors of cells depend on the structure of the template. The suitable tri-dimensional structure of dermis facilitates normal cell cycling. The more the structure of dermis closed to its physiological status, the better the biological behaviors of cells act. Moreover, the integrity as well as the continuity of dermal tissue is the prerequisite for serving as a template. The damage to the integrity and the continuity of dermal tissue may be one of the key reasons to lead abnormal tissue repair and scar formation. Thus, we hypothesize that the loss of dermal template may be one of the mechanism of abnormal scar formation and propose the theory of extracellular matrix framework deficiency or destruction.

[Influence of L-arginine supplementation on the plasma amino acid spectrum in burn patients].

OBJECTIVE: To explore the influence of L-arginine supplementation on the plasma amino acid spectrum in burn patients. METHODS: Ten burn patients were randomly divided into burn control (n = 5, with compound 14 amino acid injection accounting for 2% of the total caloric value), and experimental (n = 5, with intravenous injection of L-arginine which accounted for 2% of total caloric value) groups. The intake of other nutrients for these two groups of patients was the same. The nutrient regimen was begun on the 3 PBD, with one quarter of the daily supply. On 4 and 5 PBD, one half of the daily supply was given, and from 6 to 21 PBD full supplementation was given. Venous blood samples were collected on 3, 7, 14, 21 and 28 PBD for the determination of plasma levels of amino acids. Ten normal volunteers served as normal control. RESULTS: The plasma level of citrulline in both groups was significantly lower than normal value (P < 0.05) on 3 PBD before L-arginine supplementation. There was no obvious difference in plasma levels of ornithine and arginine in the two groups on 3 PBD compared with normal value (P > 0.05). The plasma level of ornithine, citrulline and arginine in burn control group declined on 3 PBD. The plasma level of arginine in experimental group on 14, 21 and 28 PBD were 280 +/- 121 micromol/L, 223 +/- 106 micromol/L and 110 +/- 44 micromol/L, respectively, which were significantly higher than those in burn control group (124 +/- 21 micromol/L, 59 +/- 15 micromol/L, 50 +/- 26 micromol/L). The plasma level of ornithine (30 +/- 5 micromol/L) and citrulline (162 +/- 44 micromol/L) on 21 PBD in experimental group were markedly higher than those in burn control group (8 +/- 7 micromol/L, 66 +/- 4 micromol/L, P < 0.05 or 0.01). There was no difference in the plasma levels of other amino acids at all postburn time points between the two groups (P > 0.05). CONCLUSION: The production process of L-arginine from citrulline was accelerated after burns. The plasma levels of L-arginine, ornithine and citrulline were increased markedly after L-arginine supplementation, while that of other amino acids was not influenced. The pharmacological effects of L-arginine may be related to the promotion of ornithine cycle.

[Influence of tangential excision within 24 postburn hours on the local wound inflammatory response in patients with deep partial thickness burn].

OBJECTIVE: To investigate the influence of early tangential excision within 24 postburn hours on local wound inflammatory response and tissue injury in patients with deep partial thickness burn. METHODS: Twelve patients with deep partial thickness burn were enrolled in the study. Tangential excision was performed within 24 postburn hours. The same wound of a patient was divided into three areas for harvesting tissue samples, i.e. pre-operation, post-operation and non-operation areas. For each patient, the biopsies from the pre-operation area were harvested before tangential excision, while those from post-operation and non-operation areas were harvested during 5 to 7 postburn days (PBDs). The contents of IL-8, MPO and MDA in wound tissue were determined by tissue culture and chemical colorimetry, respectively. HE and Mason's staining were employed to assess the degree of necrosis of the wound tissue. RESULTS: The IL-8, MPO and MDA contents in the local wound tissue after tangential excision were 6.83 +/- 1.85 microg/L, 4.07 +/- 0.87 U/g, and 8.94 +/- 5.66 micromol/g, respectively, which were significantly lower than those from non-operation area (P < 0.01). The inflammatory response in the non-operative wound area was distinct with expansion of necrotic tissue area. In contrast, the local inflammatory response of the wound after tangential excision was ameliorated without the enlargement of necrotic tissue area. CONCLUSION: Tangential excision within 24 postburn hours could be beneficial in ameliorating local inflammatory response, and in preventing progressive deepening of the burn wounds, thus it could accelerate wound healing in patients with deep partial thickness burn.

[An experimental study on the effects of postburn dietary supplementation of enhanced nutrients].

OBJECTIVE: To evaluate the effects of postburn dietary supplementation of arginine (Arg), omega-3 polyunsaturated fatty acid (omega-3PUFA) and glutamine (Glu) on the metabolism, immunology and wound healing in scalded rats. METHODS: Thirty Sprague-Dawley (SD) rats inflicted with 30% total body surface area deep partial thickness scald on the back after the gastrostomy catheter was placed were employed as the model. The rats were randomly divided into A and B groups, and all of them received continuous isonitrogenous (25% protein, 12% fat, 63% carbohydrate), isocaloric (175 kcal/kg/day), and isovolemic intragastric tube feedings. The contents of Arg, omega-3PUFA, Glu in the dietary of B group were enriched. The parameters were measured on the 10th day after injury, including the response of spleen cells to ConA, the plasma levels of PGE(2), IL-2, albumin, transferrin, glucagons, cortisol in blood, the urinary content of vanillylmandelic acid (VAM) in 24-hour urine, the content of hydroxyproline, the ratio of type I to type III collagen in burn wounds, and the nitrogen content in the liver and in the jejunal mucosa, as well as the weight changes, skin delayed hypersensitivity test, and wound healing time. RESULTS: It was revealed that the serum level of albumin, the nitrogen content in the liver and in the jejunal mucosa were obviously higher in B than those in A group. At the same time, there was no statistical difference in the plasma levels of cortisol and glucagons and urinary content of VAM between the two groups, nor in body weight changes. Meanwhile, the response of spleen cells to ConA and the skin delayed hypersensitivity induced by DNFB 14 days after injury in group B were also enhanced compared with those in group A. Although the expression of PGE2 from peritoneal macrophages was lower, the content of hydroxyproline from burn wounds in group B was significantly higher than that in group A, and the ratio of type l to type III collagen in group B was significantly lower than that in group A. Compared with group A, the wound healing time in group B was significantly shortened (P < 0.05). CONCLUSION: The low-fat and high-protein feeding diet with enriched arginine, omega-3 PUFA, glutamine could benefit the nutritional status after burn injury, thus improve the immunological function and promote wound healing.

[Establishment of an animal model with hypertrophic scar].

OBJECTIVE: To establish an animal model of hypertrophic scar on the rabbit ears and to observe the dynamic process of scar formation on the ventral side of the rabbit ears. METHODS: Full skin defect (2 cm x 5 cm in diameter) was created on the ventral side of 60 ears in 32 New Zealand white rabbits. 1% silver sulfadiazine cream was applied to the wounds, and the dressing was changed once a week till wound healing. Four ears without operation were taken as control. Wound healing under natural condition was observed continuously for 12 months after the operation. The scar proliferation on the rabbit ears was observed by light microscope and transmission electron microscope, and the scar index was determined by computer image analyzing system. RESULTS: The color, thickness and texture of the wounds on the rabbit ears after epithelization underwent a process of scar overgrowth, maturation and degeneration. The change in scar index was in accordance with the wax and wane of scar proliferation. CONCLUSION: The hypertrophic scar formed on the ventral side of rabbit ears after natural healing of full thickness skin wounds was similar to that in humans. The results indicated that this animal model was ideal for the study of the developmental mechanism of hypertrophic scars and for the evaluation of the efficacy of the prevention and treatment of hypertrophic scars.