SPF Rats Research Articles

Housing RCS rats under specific pathogen-free conditions mildly ameliorates retinal degeneration and alters intestine microbiota

Retinitis pigmentosa (RP) is a genetic blinding disease with over 80 causative genes. Disease progression varies between patients with similar genetic backgrounds. We assessed the association between environment, gut microbiota, and retinal degeneration in the RP rat model Royal College of Surgeons (RCS). The rats were born and raised for two generations under specific pathogen-free (SPF, n = 69) or non-SPF conditions (n = 48). At the age of four weeks, SPF rats had significantly shorter dark-adapted a-wave and dark and light-adapted b-wave implicit times by electroretinogram (p = 0.014, p = 9.5*10−6, p = 0.009, respectively). The SPF rats had significantly less photoreceptor apoptosis at ages four, eight, and twelve weeks (all p < 0.022), significantly thicker debris zone at age 14 weeks, and smaller hypofluorescent lesions in SPF rats at ages 10–16 weeks, especially in the inferior retina. The non-SPF rats had significantly higher microbiota alpha diversity (p = 0.037) and failed to present the age-related maturation of Proteobacteria, Spirochaetes, Actinobacteria, and Bacteroidetes seen in SPF conditions. Specific microbial amplicon sequence variants were reduced in rats with more severe retinal degeneration. Our data suggest an environmental effect on retinal deterioration in RCS rats. These findings may lead to the development of novel microbiome-related interventions for retinal degeneration.

Effects of simulated gas of thermobaric bomb charge explosion on Tau protein phosphorylation and cognitive function in rats

Objective: To explore the effect of simulated gas of thermobaric bomb charge explosion on cognitive function and the related mechanism of damage. Methods: In January 2022, thirty-two SPF rats were selected and randomly divided into control group, exposed group 1, 2 and 3 (the exposure time of the simulated gas of the explosion of the thermobaric bomb charge was 5 min, 10 min and 15 min, respectively) according to random number table method, with 8 rats in each group. The simulated gas of the explosion of the thermobaric bomb charge were CO 0.15%, CO(2) 3%, NO 0.1%, O(2) 15%, and the rest were N(2). After 30 days of exposure, water maze was used to detect the learning and memory function of rats. Golgi staining was used to observe the number distribution and morphological structure of hippocampal neurons in rats. Western blot was used to detect the expression of Tau-5, pSer262, pSer396, pThr181 and pThr231 proteins in rats. Repeated measure ANOVA was used to compare the design data of repeated measure, one-way ANOVA was used for multi-group mean comparison, and LSD method was used for pound-wise comparison. Results: There were significant differences in the results of repeated measurement ANOVA of the water maze localization navigation test (F=80.98, P<0.001), and there was an interaction between the group and the training days (F=2.16, P=0.022). There were significant differences in escape latency of rats at the 2nd, 3rd, 4th and 5th days among all groups (P<0.05). The results of spatial exploration showed that the frequency of rats crossing the platform was significantly different among all groups (F=4.49, P=0.011). The frequency of rats crossing the platform in exposed group 2 and exposed group 3 was lower than that in control group, and the frequency of rats crossing the platform in exposed group 3 was lower than that in exposed group 1 (P<0.05). With the increase of exposure time, the number of hippocampal neurons decreased, and the dendrite spine density of neurons in CA1 region decreased (P<0.05). Compared with the control group, there was no significant difference in the relative expression level of Tau-5 protein in all exposed groups (P>0.05), but the expression level of pSer262 protein was significantly increased (P<0.05). Compared with the control group, the protein expressions of pSer396, pThr181 and pThr231 in exposed group 2 and exposed group 3 were significantly increased (P<0.05) . Conclusion: The simulated gas of the explosion of the thermobaric bomb charge may contribute to the development of cognitive dysfunction by damaging hippocampal neurons with aberrant phosphorylation of Tau proteins.

Mechanism of Huangqin Qingre Chubi Capsules in improving rheumatoid arthritis based on METTL3-SFRP4/Wnt/β-catenin pathway

The effect and mechanism of Huangqin Qingre Chubi Capsules(HQC) on rheumatoid arthritis(RA) were studied.Seventy male SPF rats were randomly divided into normal group, model group, low-(0. 18 g·kg~(-1)), middle-(0. 36 g·kg~(-1)), and high-(0. 72 g·kg~(-1)) dose groups of HQC, methotrexate group(MTX, 0. 75 mg·kg~(-1)), and negative control group(NC group, model +saline). Adjuvant arthritis fibroblast-like synoviocytes(AA-FLS) were divided into normal group, model group, low-, middle-, and high-dose groups of HQC, and negative control group. RT-qPCR and Western blot were used to detect the m RNA and protein expressions of METTL3, SFRP4, β-catenin, CCND1, c-Myc, MMP3, and fibronectin. The protein expression of MMP3 and β-catenin was detected by immunofluorescence. The gene expression level of METTL3 on AA-FLS was knocked down to further examine the expression of each gene. ELISA measured the levels of IL-1β, IL-6, and IL-8. The results showed that compared with the normal group, rats in the model group found redness and swelling in their limbs and significantly increased joint swelling. Compared with the model group, the joint swelling degree of each treatment group significantly decreased(P&lt;0. 05). The paw retraction threshold and body weight mass index both significantly increased(P&lt;0. 05). METTL3 was highly expressed on AA and negatively correlated with the expression of SFRP4. After treatment, the m RNA and protein expression of METTL3, β-catenin, CCND1, c-Myc, fibronectin, and MMP3 were significantly decreased on AA-FLS(P&lt; 0. 05). Compared with the model group, knocking down METTL3 resulted in reduced m RNA and protein expression of β-catenin, CCND1, c-Myc, fibronectin, and MMP3(P&lt; 0. 05). At the same time, the m RNA and protein expressions of β-catenin, CCND1, c-Myc, fibronectin, and MMP3 in the HQC+METTL3 knockdown group were significantly lower than those in the METTL3 knockdown group(P&lt;0. 05). HQC could reduce the levels of IL-1β, IL-6, and IL-8 to varying degrees(P&lt;0. 05). The results indicate that HQC has a significant improvement effect on arthritis in AA rats. The expression of METTL3 is significantly increased in synovial tissue and AA-FLS of AA rats, which may be a potential target for the diagnosis and treatment of RA. HQC improves RA through the METTL3-SFRP4/Wnt/β-catenin signaling pathway and has significant antiinflammatory and anti-rheumatic effects.

"Simmer pus and grow flesh" method promotes chronic wound healing in rats via bFGF-Wnt β-catenin signaling pathway.

The purpose of this study was to explore the mechanism of "simmer pus and grow meat" method based on bFGF regulating WNT / β-Catenin signaling pathway. Of 100 SPF rats, 25 were randomly selected as blank group, and 75 rats were established chronic infectious wound model and divided into blank group, model group (normal saline treatment, n = 25), experimental group (purple and white ointment treatment, n = 25), and wet burn ointment group (wet burn treatment, n = 25). The wound healing rate of rats was compared. The protein expressions of PCAN, VEGF, bFGF, β-Catenin, GSK-3β and C-Myc in granulation tissues were detected. On the 7th day, the wound healing rate of the model group was lower than that of the other 3 groups (P<0.05), and the wound healing rate of the positive control group was higher than that of the experimental group and the control group (P<0.05). The expressions of bFGF, GSK-3β and C-MyC in model group were higher than those in control group (P<0.05). The β-catenin protein expression in the model group was lower than that in the control group (P<0.05), and the β-catenin protein expression in the experimental group and the positive control group was higher than that in the model group (P<0.05). The expressions of PCAN and VEGF in model group were lower than those in model group (P<0.05). We found that Zibai ointment promotes chronic wound healing by modulating the bFGF/Wnt/β-Catenin signaling pathway.

Effects of Desmodium caudatum on Gastrointestinal Hormones and Intestinal Flora in Rats with Gastritis.

In order to preliminarily explore the effects of Desmodium caudatum on gastritis and intestinal flora in rats, a chronic gastritis rat model was established using the classic sodium salicylate method. Eighteen SPF rats were divided into three groups: the control group (Group C), the model group (Group M), and the treatment group (Group T). Pathological sections of the gastric wall were taken from rats in each group. Furthermore, the concentrations of gastrin and malondialdehyde in the serum of rats in each group were determined by ELISA. Additionally, the effects of D. caudatum on the intestinal flora of rats with gastritis were explored through a detailed comparison of gut bacterial communities in the three groups, employing Illumina-based 16S rRNA gene sequencing. The results indicated that D. caudatum decoction could reduce the malondialdehyde content and increase the gastrin content. Moreover, D. caudatum decoction was found to enhance the diversity and abundance of intestinal flora, exerting a positive impact on the treatment of gastritis by regulating and restoring the intestinal flora.

Effect of nerve growth factor on elderly degenerative knee osteoarthritis pain

To explore effect of nerve growth factor (NGF) antibody on knee osteoarthritis (KOA) pain model was evaluated by in vitro model. Thirty male SPF rats aged 28-week-old were divided into blank group (10 rats with anesthesia only). The other 20 rats were with monoiodoacetate (MIA) on the right knee joint to establish pain model of OA, and were randomly divided into control group (injected intraperitoneal injection of normal saline) and treatment group (injected anti-NGF) intraperitoneal after successful modeling, and 10 rats in each group. All rats were received retrograde injection of fluorogold (FG) into the right knee joint. Gait was assessed using catwalk gait analysis system before treatment, 1 and 2 weeks after treatment. Three weeks after treatment, right dorsal root ganglia (DRG) were excised on L4-L6 level, immunostained for calcitonin gene-related peptide (CGRP), and the number of DRGS was counted. In terms of gait analysis using cat track system, duty cycle, swing speed and print area ratio in control and treatment group were significantly reduced compared with blank group (P<0.05). Compared with control group, duty cycle and swing speed of treatment group were significantly improved (P<0.05), and there was no significant difference in print area ratio between treatment group and blank group (P>0.05). The number of FG-labeled DRG neurons in control group was significantly higher than that in treatment group and blank group (P<0.05). The expression of CGRP in control group was up-regulated, and differences were statistically significant compared with treatment group (P<0.05). Intraperitoneal injection of anti-NGF antibody inhibited gait injury and upregulation of CGRP in DRG neurons. The results suggest that anti-nerve growth factor therapy may be of value in treating knee pain. NGF may be an important target for the treatment of knee OA pain.

Gallic acid alleviates lipopolysaccharide-induced renal injury in rats by inhibiting cell pro-death and inflammatory response and its mechanism.

The mechanism of gallic acid in improving lipopolysaccharide-induced renal injury in rats was investigated by studying the pro-death and inflammatory response of cells. SPF rats were randomly divided into 4 groups with n=10 in each group. Blank control group: normal saline injection; The model group was injected with LPS induced model (LPS group); Low dose gallic acid group (LPS+L-GA group); Middle dose gallic acid group (LPS+M-GA group). The expression of serum inflammatory factors IL-1, IL-1β, IL-18, and MCP-1 were detected by Elisa. Western blot assay was used to detect the expression of inflammation-related proteins. The contents of BUN, Scr, SUA, Serum cystatinALB, and ACR were determined by the biochemical analyzer. The pathological tissue sections were used to observe the kidney injury in each group. The renal expressions of NLRP3, Caspase-1, GSDMD, and IL-1β were detected by immunohistochemistry. The activation of the AMPK/SIRT1 signaling pathway was detected by Western blot assay. The LPS-induced mouse kidney injury model was established successfully. Compared with the model group, different doses of gallic acid can improve the expression of renal biochemical indexes (P<0.05); At the same time, gallic acid can activate AMPK/SIRT1 and reduce kidney injury in mice (P<0.05); Compared with the model group, the expression of pyroptosis gene, the expression of genes related to inflammatory factors and the expression of inflammatory factors were decreased in the gallic acid injection group (P<0.05). By activating the AMPK/SIRT1 signaling pathway, gallic acid can inhibit the scorch death and validation effect in mice, thereby protecting the kidneys of mice.

CSF - injected contrast medium enhances post-traumatic spinal cord cysts. An experimental study in rats.

Spinal cord injury (SCI) is still one of the most challenging problems in neurosurgical practice. One of the major obstacles to neural regeneration following trauma is the formation of glial scarring and post-traumatic cysts which acts against proper growth of axons through the site of injury. Cerebrospinal fluid (CSF) delivery of bioactive agents into cystic cavities could represent a promising therapeutic strategy. In the present study, we investigated specifically the dynamics of intradural delivery of contrast medium and its relocation into post-traumatic cysts in an experimental model of spinal cord cryoinjury in rats. 32 male Sprague Dawley SPF rats were submitted to injury as previously described. Omnipaque-240 was injected either into the cisterna magna or at the level of the cauda equina. Subsequently, cerebral CT scan examinations were performed in order to check the CSF dynamics of the contrast medium. There was a steady accumulation of contrast medium into post-traumatic cysts as early as five minutes after injection. A dosage of 65 mg of iodine per kilogram ensured an adequate feeling of the cysts at an average of 30 minutes. Our data indicate that intraspinal injection of bioactive agents can easily reach the site of injury and fill post-traumatic cysts. This could represent an interesting potential therapeutic protocol for SCI.

Effect of high-copper diet on transference of bla CTX-M genes among Escherichia coli strains in rats' intestine.

Both ceftiofur (CTO) and high copper are widely utilized in animal production in China, and the occurrence of CTX-M-carrying Escherichia coli in food-producing animals is increasing. There are some specific associations between in-feed high-level copper and antibiotic resistance, but research in Gram-negative bacteria such as E. coli remains scarce. This study aimed to evaluate the effect of high-copper diet on the horizontal transfer of bla CTX-M-1 among E. coli. A total of 32 male SPF rats aged 21 days were randomly assigned to the following four groups: control (6 mg/kg in-feed copper, C-), high copper (240 mg/kg in-feed copper, H-), CTO (6 mg/kg in-feed copper with oral CTO administration, C+), and high copper plus CTO (240 mg/kg in-feed copper with oral CTO administration, H+). All rats were orally inoculated with an E. coli strain harboring a conjugative plasmid carrying bla CTX-M-1, and the C+ and H+ groups were given 10 mg/kg of body weight (BW) CTO hydrochloride at 26, 27, and 28 days, while the C- and H- groups were given salad oil at the same dose. Fecal samples collected at different time points were used for the enumeration of E. coli on Mac plates or for molecular analysis using PCR, pulsed-field gel electrophoresis (PFGE), S1-PFGE, and Southern-blot hybridization. The results showed that the number of the bla CTX-M-1 gene in the H- group was higher and that the loss speed of this gene was slower compared with the C- group. After administration of CTO, the counts of cefotaxime-resistant E. coli were significantly higher in the C+ group than that in the corresponding control group (C+ vs. C-; H+ vs. H-). In the in vitro test, the results showed that the transfer rates of the conjugation induced by the H- (12 mmol/L) group were significantly higher than that of low copper (2 mmol/L) group. The indigenous sensitive isolates, which were homologous to the bla CTX-M-positive isolates of rat feces, were found by PFGE. The further analysis of S1-PFGE and Southern-blot hybridization confirmed that the bla CTX-M-1 gene in new transconjugants was derived from the inoculated strain. Taken together, high-copper diet facilitates the horizontal transfer and maintenance of the resistant genes in the intestine of rats, although the effects of antibiotics on bacterial resistance appearance and maintenance are more obvious.

Effect of moxibustion on immune function homeostasis in rats with diarrhea irritable bowel syndrome based on SCF/c-kit signaling pathway

To observe the effects of moxibustion on the stem cell factor (SCF)/tyrosine kinase receptor (c-kit) signaling pathway and immune function in rats with diarrhea irritable bowel syndrome (IBS-D), and to explore the mechanism of moxibustion for IBS-D. Among 52 young rats born from 6 healthy pregnant SPF rats, 12 rats were randomly selected into the normal group, and the remaining 40 rats were treated with the three-factor combination method of maternal separation, acetic acid enema and chronic restraint stress to establish the IBS-D rat model. Thirty-six rats with successful IBS-D model were randomly divided into a model group, a moxibustion group, and a medication group, 12 rats in each group. The rats in the moxibustion group were treated with suspension moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the medication group were treated with intragastric administration of rifaximin suspension (150 mg/kg). All the treatments were given once a day for 7 consecutive days. The body mass, loose stool rate (LSR), the minimum volume threshold when abdominal withdrawal reflex (AWR) scored 3 were measured before acetic acid enema (35 days old), after modeling (45 days old), and after intervention (53 days old). After intervention (53 days old), HE staining was used to observe the morphology of colon tissue, and spleen and thymus coefficients were measured; ELISA method was used to detect serum inflammatory factors (tumor necrosis factor a [TNF-a], interleukin [IL]-10, IL-8), T-lymphocyte subsets (CD+4, CD+8, CD+45), value of CD+4/CD+8 and immune globulin (IgA, IgG, IgM); real-time PCR method and Western blot method was used to detect the expression of SCF, c-kit mRNA and protein in colon tissue; immunofluorescence staining method were used to detect positive expression of SCF and c-kit. After intervention, compared with the normal group, in the model group, the body mass and the minimum volume threshold when AWR scored 3 were decreased (P<0.01), LSR, spleen and thymus coefficients, serum levels of TNF-α, IL-8, CD+4, CD+45, CD+4/CD+8, IgA, IgG, IgM were increased (P<0.01), serum IL-10 level and protein and mRNA expression of SCF and c-kit in colon tissue were decreased (P<0.01), and the positive expression of SCF and c-kit was decreased (P<0.01). Compared with the model group, in the moxibustion group and the medication group, the body mass and the minimum volume threshold when AWR scored 3 were increased (P<0.01, P<0.05), LSR, spleen and thymus coefficients, serum levels of TNF-α, IL-8, CD+4, CD+8, CD+45, CD+4/CD+8, IgA, IgG, IgM were decreased (P<0.01, P<0.05), serum IL-10 level and protein and mRNA expression of SCF and c-kit in colon tissue were increased (P<0.01), and the positive expression of SCF and c-kit was increased (P<0.01). Compared with the medication group, in the moxibustion group, the level of serum CD+4 was decreased (P<0.05), the value of CD+4/CD+8 was increased (P<0.01), and there was no significant difference in other indexes (P>0.05). The expression of SCF and c-kit mRNA was positively correlated with the minimum volume threshold when AWR scored 3 and IL-10 (P<0.01), and negatively correlated with remaining indexes (P<0.01, P<0.05). Moxibustion could reduce visceral hypersensitivity, improve symptoms of abdominal pain and diarrhea in IBS-D rats, and its mechanism may be related to up-regulation of the expression of SCF/c-kit signaling pathway and improvement of IBS-D immune function.

Detection of Methicillin-Resistant Staphylococcus aureus in Clinical and Subclinical Mastitis in Ruminants and Studying the Effect of Novel Green Synthetized Nanoparticles as One of the Alternative Treatments.

Mastitis is an important disease in dairy animals worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of clinical and subclinical intramammary infections. In the current study, we isolated bacteria from 150 mastitic milk samples. Multiplex PCR was used to detect the methicillin resistance genes in S. aureus to detect the occurrence of MRSA isolates. Green synthesized titanium dioxide nanoparticles (TiO2 NPs) using aqueous leaf extracts of Artemisia herb Alba (A. herb Alba TiO2 NPs). The antibacterial efficacy of these nanoparticles was evaluated (in vitro and in vivo) against collected MRSA isolates using the disc diffusion method and SPF rats. Out of 150 mastitic milk samples, the frequency of S. aureus was 38 (25.3%), that of E. coli was 45 (30%), that of Klebsiella spp. Was 7 (4.7%), and that of Streptococcus spp. Was 11 (7.3%). Among 38 positive isolates of S. aureus, MRSA was 16 (42.1%) by antimicrobial sensitivity testing (AST) and 14 (38.8%) by multiplex PCR. The MRSA isolates were shown to have 100% resistance to penicillin and methicillin, 87.5% resistance to gentamicin, 50% resistance to cefoxitin and amoxicillin, and 75% resistance to ampicillin and ampicillin/sublactam with low resistance against erythromycin, ciprofloxacin, tetracycline, and levofloxacin by AST, respectively. A. herb Alba TiO2 NP formation was observed by changing the colour from white to dark green. The UV spectrum revealed absorbance peaks at 240-250 nm, and their sizes ranged from 42-66 nm and 11 to 45 nm by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). ‏A. herb Alba TiO2 NP suspensions were evaluated against MRSA, with the highest zone of inhibition (43 ± 0.45 mm) at a concentration of 40 μg/ml. Hematological parameters and histological examination after oral administration of 20 mg/kg of A. herb Alba TiO2 NPs indicated that A. herb Alba TiO2 NPs can be used as a new antimicrobial against resistant bacteria (MRSA) with consideration of the dose and methods of synthesis of plant-based compounds.

Pressing Intervention Promotes the Skeletal Muscle Repair of Traumatic Myofascial Trigger Points in Rats.

ObjectiveTo observe the effect of pressing intervention on the skeletal muscle repair of myofascial trigger points (MTrPs) in rats and explore the mechanism of pressing intervention on the deactivation of trigger points.MethodsThirty SPF rats were randomly divided into blank group, model group and press group, with 10 rats in each group. The MTrPs models were established by blunt striking plus eccentric exercise, and then evaluated. The press group was given a pressing intervention with a self-made device for 14 days, and the rats in the other two groups were fed normally. Soft tissue tension (STT) D0.2 and pressure pain threshold (PPT) were measured before and after intervention. The skeletal muscle tissue at MTrPs was extracted and assessed by hematoxylin–eosin (HE) and Masson staining. The expression of collagen I, collagen III, α- smooth muscle actin (α-SMA), myosin heavy chain (MHC) and fibronectin (FN) were detected by Western Blotting. Enzyme linked immunosorbent assay (ELISA) was used to evaluate the expression of substance P (SP), 5-hydroxytryptamine (5-HT), cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2).Results(1) Compared with the blank group, the PPT and D0.2 reduced (P < 0.05) in the model group; while compared with the model group, the PPT and D0.2 increased (P < 0.05) in the press group. (2) Compared with the blank group, the model group showed obvious spontaneous potentials with higher amplitude and frequency, which were also much higher than those of the press group (P < 0.05). (3) The HE and Masson staining results showed evident fibrosis in the muscle tissue of the model group, with a larger area of collagen fibers relative to that of the press group (P < 0.05). (4) The amount of collagen I, collagen III, FN, α- SMA, SP, 5-HT, COX-2 and PGE2 increased and the content of MHC decreased (P < 0.05) in the model group, as compared to the blank group; while all the substances (P < 0.05), instead of MHC which increased (P < 0.05), in the press group were decreased as compared to the model group.ConclusionPressing intervention on the MTrPs in rats can alleviate chronic inflammation, inhibit fibrosis, promote skeletal muscle repair and relieve pain.

Effect of leech-centipede medicine on improving erectile function in DIED rats via PKC signalling pathway-related molecules

Ethnopharmacological relevanceLeeches (pinyin name Shui Zhi; Latin scientific name Hirudo; Hirudinea; Hirudinidae) and centipedes (pinyin name Wu Gong; Latin scientific name Scolopendridae; Chilopoda; Scolopendridae) are traditional Chinese medicines, and they belong to the family entomology. A combination of leech and centipede is used as an effective medicine to promote blood circulation and remove blood stasis in traditional Chinese medicine, and “leech-centipede” medicine has been used in many prescriptions to treat diabetic vascular disease, including diabetic erectile dysfunction (DIED). However, its specific mechanism remains unclear and requires in-depth study. Aim of the studyThis study aimed to investigate the mechanism of “leech-centipede” medicine to improve erectile dysfunction-associated diabetes by detecting PKC pathway-related molecules. Materials and methodsThe active ingredients of “leech-centipede” medicine were identified using high performance liquid chromatography (HPLC). Fifty male SPF rats were injected with streptozotocin to induce the DM model. Eight weeks later, the DMED model was validated with apomorphine. The DIED rats were divided into five groups—T,P,DD,DZ, and DG—and were separately treated with tadalafil, pathway inhibitor LY333531 and low-, medium-, and high-dose “leech-centipede” medicine for 8 weeks. After treatment, the blood glucose level was measured, erectile function with apomorphine was assessed, the LOX-1, sE-selectin, sICAM-1, SOD, and MDA in serum was evaluated by enzyme-linked immunosorbent assay, and flow cytometry was performed. After the collection of penile tissue, the related protein and mRNA expression was assessed by Western blotting and PCR, and the tissue and ultrastructure were analysed by HE staining, immunohistochemistry and scanning electron microscopy. ResultsAfter treatment, the erectile function of rats was significantly improved in the T,P,DD,DZ, and DG groups compared with that in the model group. Thus, “leech-centipede” medicine can significantly reduce the levels of LOX-1, sE-selectin, sICAM-1, EMPs and CD62P to protect vascular endothelial function and anti-platelet activation, improving DIED rat erectile function. Additionally, “leech-centipede” medicine can increase SOD expression and decrease MDA expression, reducing the possibility of oxidative stress injury in DIED rats and improving the antioxidant capacity. Moreover, “leech-centipede” therapy can dramatically reduce the protein and mRNA expression of DAG, PKCβ, NF-κB, and ICAM-1, improve vascular endothelial injury in DIED rats and inhibit abnormal platelet activation. Conclusion“leech-centipede” medicine can improve erectile dysfunction by inhibiting the expression of PKC pathway-related molecules in DIED rats and protects endothelial function and anti-platelet activation.

Synthesis of Novel Biomaterial for the Treatment of Sepsis in Rats

In this research, glutamine (GLN) and L-Tyrosine were synthesized into AL-GLN synthetic biomaterials to explore their protective effect on the myocardium of sepsis symptoms. In the process of preparation, acyl chloride method was used to generate, which was generated by acylation reaction and ammonolysis reaction. In the ammonolysis reaction, the original ammonia water reaction was replaced by the ammonia water/dimethylformamide (DMF) system. The biomaterial was characterized, and it was confirmed that the product recovery rate could be maximized by adjusting the pH value to 9 during crystallization stage, and the purity could be increased from 94.23% to 98.45% after some technological improvement. During the experiment of myocardial protection in sepsis, 60 SPF rats were selected for the experiment and divided into three groups, namely the unmodified surgery group, the GLN group and the AL-GLN group. Rats with sepsis were obtained by cecum ligation and perforation. The myocardial apoptosis index and the gene expression of Bcl-2/p53mRNA were detected at 6 h, 12 h and 24 h after operation in different groups. The results showed that the expression of Bcl-2mRNA in the surgical group was significantly down-regulated compared with that in the surgical group (P &lt; 0.05), the expression of Bcl-2mRNA in the GLN group was significantly increased compared with that in the surgical group (P &lt; 0.05), and the expression of p53mRNA in the AL-GLN group was first decreased and then slowly increased compared with that in the surgical group (P &lt; 0.05).

Study on the differences between moxibustion at different points in the effects on the anti-fatigue ability of rats undergoing one-time exhaustive swimming

To observe the effects of moxibustion at Shenshu (BL 23), Zusanli (ST 36) and Shenque (CV 8) on the energy metabolism and endocrine metabolism indicators of rats undergoing one-time exhaustive swimming, and to explore the differences between moxibustion at different points in the effects on anti-exercise fatigue. Forty-eight male SPF rats were randomly divided into a blank group, a model group, a non-meridian and non-acupoint group, a Shenshu (BL 23) group, a Zusanli (ST 36) group, and a Shenque (CV 8) group using random number table method, with eight rats in each group. Except for the blank group, rats in the other groups were subjected to replicating the one-time exhaustive model using the weight-bearing swimming experiment. Except for the model group, the other model rats received mild moxibustion immediately after swimming. Rats in the non-meridian and non-acupoint group received mild moxibustion at bilateral subcostal non-meridian and non-acupoint points, those in the Shenshu (BL 23) group received mild moxibustion at bilateral Shenshu (BL 23), those in the Zusanli (ST 36) group received mild moxibustion at bilateral Zusanli (ST 36), and those in the Shenque (CV 8) group received mild moxibustion at Shenque (CV 8) for 15 min. Four hours after the exhaustive swimming, femoral artery blood was collected to detect blood lactate (BLA), lactate dehydrogenase (LDH), creatine kinase (CK), creatinine (CRE), blood urea nitrogen (BUN), cortisol (C) and testosterone (T) levels, and calculate the T/C ratio. Compared with the blank group, rat’s serum levels of BLA, LDH, CK, BUN and C in the model group and the non-meridian and non-acupoint group were increased, and serum levels of CRE and T, and T/C ratios were decreased (P<0.01 or P<0.05); compared with the model group and the non-meridian and non-acupoint group, the serum levels of BLA, LDH, CK, BUN and C in the Shenshu (BL 23) group, Zusanli (ST 36) group and Shenque (CV 8) group were decreased, and the serum CRE and T levels, and the T/C ratios were increased (all P<0.01); compared with the Shenshu (BL 23) group, the serum CK level was decreased in the Shenque (CV 8) group (P<0.01), the serum levels of T and C were decreased in the Zusanli (ST 36) group and Shenque (CV 8) group (P<0.01 or P<0.05), and the T/C ratio was increased in the Shenque (CV 8) group (P<0.01); compared with the Zusanli (ST 36) group, the serum CK and BUN levels were decreased (P<0.01, P<0.05), and the T/C ratio was increased in the Shenque (CV 8) group (P<0.05). Moxibustion at Shenshu (BL 23), Zusanli (ST 36) and Shenque (CV 8) shows different anti-fatigue effects by regulating the energy metabolism and endocrine metabolism in rats undergoing one-time exhaustive swimming. Moxibustion at Shenshu (BL 23) is better in promoting energy synthesis. Moxibustion at Shenque (CV 8) is more effective in regulating synthesis and decomposition of the skeletal muscle proteins.

Research on the Effects of the Extract of Polygala fallax Hemsl on Sex Hormones and ?-EP in Perimenopausal Rat Models

Objective: To study the effects of the ethnic medicine Polygala fallax Hemsl with Guangxi characteristics on the sex hormones and ?-EP in research objective perimenopausal rat models. Methods: 40 female SPF rats were randomly divided into 4 groups, including the normal, model, high-dose and low-dose groups. Rats of three groups except for the normal one were treated with perimenopausal modelling through the method of subcutaneous injection of compound 4-VCD for 15 consecutive days. Rats of the normal and model group were normally fed without any treatment. Rats of the high-dose and low-dose groups were administered by high- and low-dose intragastric administration of the extract of Polygala fallax Hemsl. According to the menstrual cycle of the vaginal smear of the rat, each menstrual cycle is a course of treatment and 6 consecutive courses of treatment would be given. The indexes of serum sex hormones (E2, FSH, LH) and ?-EP of rats in each group were observed after treatment. Results: After the treatment of 6 cycles, for the levels of ?-EP and E2, the model group was lowest (P&lt;0.05), the normal group was highest (P&lt;0.05); and the high-dose group was higher than the low-dose group; For the levels of FSH and LH, the normal group was lowest (P&lt;0.05), the model group was highest (P&lt;0.05), and the high-dose group was lower than the low-dose group. Conclusion: Guangxi characteristic national medicine Polygala fallax Hemsl can effectively improve the levels of serum sex hormones and ?-EP in perimenopausal rat models and relieve the related symptoms with a certain dose-effect relationship.

Effect of High Calorie Diet on Intestinal Flora in LPS-Induced Pneumonia Rats

Intestinal flora plays an important role in inflammatory response to systemic or local organs of its host. High calorie diet has been shown to aggravate the condition of pneumonia and delay recovery, especially in children. However, the underlying mechanisms remain unclear. This study placed SPF rats in a conventional environment, high calorie diet or LPS atomization was performed respectively or combined. Analysis of high-throughput sequencing of intestinal content combined with animal weight, organ index, serum inflammatory factors indicators and bioinformatics found that after pulmonary infection combined with a high-calorie diet, rats showed significant changes such as weight loss and increased lung weight index, and their lung and intestinal tissues showed more obvious inflammatory changes. And its gut flora structure suggests, the abundance of Leuconostocaceae in significantly reduced; abundance of Staphylococcus, Planococcaceae, Staphylococcus, Staphylococcaceae, Bacillales, Gemellales and Aerococcus significant increased. The study showed that high calorie diet and LPS atomization synergistically promoted pneumonia process in rat pups, which is related to changes in structure of intestinal flora. It is worth noting that pneumonia rats fed by convention diet also causing intestinal flora imbalance.

NADPH oxidase participates in pancreatic injury in rats with acute hypertriglyceridemic pancreatitis by regulating Akt/GSK3 β pathway

Objective To investigate the aggravation of pancreatic tissue injury in rats with acute hypertriglyceridemic pancreatitis and the possible role of NADPH oxidase (NOX). Methods Thirty SPF rats were randomly (random number)divided into five groups: N group, H group, NLAP group, HLAP group and HAPO group. AMY, TG, TC and FFA levels were detected. The pathological changes of pancreas were observed under light microscope and the ultrastructural changes of pancreatic acinar cells were observed by TEM. Serum levels of MDA, SOD, IL-1β, TNF-α and LDH were detected. The expression of NOX4, p-Akt and p-GSK3β in pancreas was detected by immunofluorescence, and the expression of NF-κB and TNF-α in pancreas was detected by immunohistochemistry. Results Intraperitoneal injection of P-407 could significantly increase the levels of serum TG, TC and FFA in rats. After acute pancreatitis induced by L-Arg, the levels of serum AMY in the NLAP and HLAP groups were significantly increased, while Apocynin could significantly decrease the level of serum AMY. Compared with the NLAP group, the pathological injury of pancreatic tissue in the HLAP group was more serious, the level of inflammatory mediators was significantly increased, and the cell necrosis was more serious. After inhibiting NOX, the activation of Akt/GSK3β pathway was regulated and the pancreatic injury was improved. Conclusion In HTGP, NOX aggravates pancreatic injury by regulating the activation of Akt/GSK3 β pathway. Inhibition of NOX expression can play a protective role in pancreas injury of HTGP.. Key words: Hypertriglyceridemia; Acute pancreatitis; Pancreatic injury; NADPH oxidase; Oxidative stress

DNA Damage Levels and DNA Repair Capability of Bone Marrow‐Derived Mesenchymal Stem Cells from Sprague Dawley® SPF rats as a Quality Parameter Before Surgical Transfection for the Treatment of Gestational Diabetic Myopathy

Mesenchymal stem cells (MSCs) have an important role for regenerative medicine through their self‐renewal and differentiation capability; besides its involvement in tissue development, maintenance, and repair, being useful for skeletal muscle regeneration by the production of secretory factors with trophic and engraftment functions. We propose to use the alkaline comet assay to evaluate the DNA damage levels and DNA repair capability according to cell culture passages as one of the parameters to test the quality of MSCs before surgical transplantation to the periurethral and rectus abdominis muscle (RAM) for the treatment of gestational diabetic myopathy in female rats at postpartum. MSCs were collected from bone marrow of female Sprague Dawley® rats. The characterization of these cells was done according to the International Society for Stem Cell. After isolation using Ficoll‐Paque and washing in low glucose Dulbecco's Modified Eagle Medium (DMEM) enriched with L‐glutamine, the cells were seeded in 25 cm2 plastic flasks containing high glucose DMEM enriched with L‐glutamine plus 20% bovine fetal serum, penicillin, streptomycin and amphotericin B. The cells were harvested at 90% of confluence and subjected to comet assay analysis. The slides were incubated in alkaline electrophoresis solution for 20 minutes for DNA unwinding and the run was conducted at 25v, 0.3A and 8W for 30 minutes, at 4 °C. The slides were stained with SYBR GOLD® (1:10.000 dilution) and analyzed in a fluorescent microscope using COMET ASSAY VI software. The DNA damage levels were ranked according to the percentage of damage as follows: 1) 0 % – 5% of damage; 2) 6% – 20% of damage; 3) 21% – 40% of damage; 4) 41% –95% of damage; 5) &gt; 95% of damage. The percentage of nucleoids with such levels of damage was calculated and analyzed using the Chi‐Square test. The analysis was conducted using SPSS (21.0) and p &lt; 0.05 was considered significant. This study was approved by the local Animal Ethics Committee (1234/2017 – CEUA). The partial results demonstrated that the 5th passage has a high percentage (70.04%) of cells with high levels of DNA damage (41% – 95%) assessed by tail intensity. The proportion of cells with high percentage of DNA damage (41% – 95%) is statistically significant from the proportion of cells with low DNA damage levels (0% – 5%, 6% –20%, and 21% –40%; p &lt; 0.001). The preliminary results demonstrated that the 5th passage is not useful for surgical transplantation because of high DNA damage levels. Future experiments will be conducted to test the DNA damage of the other passages and also evaluated the effect of cell culture on DNA repair capability. Furthermore, such results will be compared with the functional evaluations of periurethral and RAM after the surgical transplantation of MSC.Support or Funding InformationSupported by São Paulo Research Foundation (2016/01743‐5)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Antiobiotic-Induced Disbiosis and Its Correction with Probiotics on Myocardial Tolerance to Ischemia-Reperfusion Injury in SPF Rats.

Changes in morphofunctional parameters of the isolated heart subjected to global ischemia-reperfusion were studied in SPF Wistar rats with antibiotic-induced dysbiosis (AID) treated with lyophilized yeast Saccharomyces boulardii and inactivated probiotic bacteria Lactobacillus reuteri KR2017. In contrast to S. boulardii, correction of dysbiosis with L. reuteri KR2017 against the background of gastric hypersecretion and standard antimicrobial therapy led to an increase in fat content and a decrease in free and bound water in tissues and to a significant reduction in myocardial infarct size caused by ischemia/ reperfusion injury.