Compressão intermitente imediata sobre anastomose vascular: Uma nova técnica para prevenir trombose? / Immediate intermittent compression over vascular anastomosis: a new technique to prevent thrombosis

Introdução: Este estudo tem o objetivo de avaliar o efeito da compressão intermitente imediata sobre anastomoses arteriais microcirúrgicas em comparação com compressão fixa e com utilização isolada de irrigação com soro fisiológico e heparina em laboratório experimental. Método: 12 ratos Wistar foram aleatoriamente divididos em três grupos para terem suas artérias femorais seccionas e anastomosadas de forma término-terminal, para comparação de patência com 30 minutos e 7 dias. Grupo I: foi realizada compressão intermitente imediata sobre a anastomose por 60 segundos; grupo II: uma compressão fixa foi mantida imediatamente após a anastomose, também por 60 segundos; grupo III, após o término da anastomose, não foi feita nenhuma intervenção adicional. Além da avaliação da patência, os animais foram pesados e medidos os diâmetros arteriais operados. Resultados: 24 artérias femorais foram abordadas. As médias de peso inicial dos ratos dos grupos I, II e III foram, respectivamente, de 243,8g, 254,6g e 260,4g, enquanto as finais foram de 264,4g, 281g e 282,1g (p<0,001). O diâmetro médio das artérias abordadas foi, respectivamente, de 0,89mm, 0,88mm e 0,90mm, e os tempos de anastomoses em minutos, de 25,6, 24,5 e 24,5, respectivamente; As patências finais após 7 dias foram, respectivamente, de 62,5% (p=0,07), 25% (p=0,48) e 50% (p=0,13). Conclusão: A compressão intermitente imediata pode ser realizada ao término de anastomoses arteriais microcirúrgicos sem prejuízo na patência final do procedimento.

Introduction: This study aims to evaluate the effect of immediate intermittent compression on microsurgical arterial anastomoses in comparison with fixed compression and only observation in an experimental laboratory. Methods: The two femoral arteries of twelve male Wistar rats were sectioned and reanastomosed to compare patency at 30 minutes and 7 days. Group I: immediate intermittent compression was performed over the anastomosis for 60 s; group II: a fixed compression was maintained immediately after the anastomosis for 60 s; group III: after completion of the anastomosis, no additional intervention was performed. In addition to the patency assessment, the animals were weighed and the operated arterial diameters were measured. Results: Twenty-four femoral arteries were examined. Initial average weights of the rats in groups I, II, and III were 243.8g, 254.6g, and 260.4g, respectively, while the final weights were 264.4g, 281g, and 282.1g (p<0.001), respectively; mean diameter of the approached arteries was 0.89, 0.88, and 0.90mm, respectively, and the anastomoses (time in minutes) were 25.6, 24.5, and 24.5, respectively; final patencies after 7 days were 62.5% (p=0.07), 25% (p=0.48), and 50% (p=0.13), respectively. Conclusion: Immediate intermittent compression can be performed at the end of microsurgical arterial anastomoses without affecting the final patency of the procedure.

Effect of echinacoside intervention on liver and pancreas injury in rats with acute pancreatitis and its mechanism / 临床肝胆病杂志

ObjectiveTo investigate the effect and mechanism of echinacoside （ECH） in improving liver injury in rats with acute pancreatitis by establishing a rat model of acute pancreatitis and liver injury. MethodsA total of 24 Sprague-Dawley rats were randomly divided into blank group （Con group）， control group （Con+ECH group）， acute pancreatitis group （AP group）， and acute pancreatitis+ECH intervention （AP+ECH group）. The rats were given intraperitoneal injection of 10 mg/kg ECH on day 7 before the establishment of the model of acute pancreatitis； at 24 hours after the last administration of cerulein， blood samples were collected via the abdominal aorta， and serum was separated for biochemical analysis including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， lactate dehydrogenase （LDH）， gamma-glutamyl transpeptidase （GGT）， alkaline phosphatase （ALP）， albumin （Alb）， total bilirubin （TBil）， cholinesterase， blood amylase （Amy）， and lipase （LPS）. HE staining was used to observe the histopathological changes of the pancreas and the liver； transmission electron microscopy （TEM） was used to observe the microstructural changes of pancreas and liver tissue； ELISA was used to measure the levels of interleukin-1β （IL-1β）， interleukin-16 （IL-6）， tumor necrosis factor-α （TNF-α）， and interleukin-10 （IL-10） in liver tissue homogenate； immunohistochemistry was used to measure the levels of TNF-α and p-p65 NF-κB in pancreas and liver tissue； Western blot was used to measure the expression levels of NF-κB pathway proteins in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the SNK test or the Dunnett’s T3 method was used for further comparison between two groups. ResultsCompared with the Con group， the AP group had significant increases in ALT， AST， GGT， LDH， ALP， TBil， Amy， and LPS （all P<0.01）， as well as significant increases in the levels of IL-1β， IL-6， IL-10， and TNF-α in liver tissue homogenate （all P<0.01）. ECH intervention reduced the levels of ALT， AST， GGT， LDH， ALP， TBil， AMY， and LPS and inhibited the secretion of IL-1β， IL-6， and TNF-α in rats with acute pancreatitis. HE staining showed that ECH intervention alleviated the vacuolar degeneration of acinar cells， inflammatory cell infiltration in pancreatic tissue， and the necrosis of hepatocytes compared with the AP group. TEM showed that compared with the AP group， there was a reduction in the degree of mitochondrial swelling in liver and pancreatic cells after ECH intervention. ECH intervention partially reversed the elevated expression levels of p-p65 NF-κB and TNF-α in liver and pancreatic tissue. In addition， the expression levels of MyD88， p-IκBα， p-IKKα， and p-p65 were upregulated in liver tissue of rats with acute pancreatitis， which could be partially reversed after ECH intervention. ConclusionEchinacoside can alleviate liver and pancreatic injury induced by acute pancreatitis by inhibiting the TLR4/MyD88/NF-κB pathway.

Effects of puerarin on maternal gestational diabetes mellitus rats and fetal growth / 中国药理学通报

Aim To observe the effects of oral puerarin (Pue) on the maternal gestational diabetes mellitus (GDM) rats and its fetal growth and development, so as to provide references for the application of Pue in the treatment of GDM. Methods The GDM rat model was established by injecting streptozotocin (STZ) into the tail vein of pregnant female rats, and the rats were treated with Pue orally for 12 days; the body weight and abortion of pregnant rats were recorded. The fasting blood glucose of pregnant rats was detected before and at the end of the treatment, and the glucose tolerance was tested on the 5th and 10th days after the administration of the drug. The cesarean sections were carried out on the 20th day of pregnancy. The blood glucose content of the fetal rats was detected, and the state of development was observed. The body weight and length were measured, as well as the placenta and the important organs weight, and the indexes of the organs were calculated. Results Compared with the model group, Pue could significantly reduce the fasting blood glucose of GDM pregnant rats and fetal rats, improve the glucose tolerance of pregnant rats, effectively alleviate the excessive weight gain of pregnant rats and overweight of fetal rats caused by GDM, and reduce the abortion rate; it could also reverse the decrease in the indexes of the organs of brain, heart, and liver, and the increase in the indexes of organs of kidney in fetal rats caused by GDM. Conclusions Pue can relieve the maternal and the fetal hyperglycemia in GDM, reduce the rate of miscarriage, reduce the incidence of macrosomia, and promote the development of vital fetal organs.

Inhibition of HSP70 release by geniposide improves angiogenesis in moist heat arthralgia spasm syndrome collagen induced arthritis rats / 中国药理学通报

Aim To investigate the relation between the effect of geniposide (GE) in improving angiogenesis in arthralgia spasm syndrome collagen induced arthritis (CIA) rats and the modulation of heat shock proteins 70 (HSP70) release. Methods A CIA model was constructed by multiple intradermal injections of complete Freund's adjuvant (CFA) and an equal volume mixture of chicken type II collagen (CCII) into the dorsal and caudal root regions of rats, on the basis of which a rheumatic fever stimulus was given to build up a moist heat arthralgia spasm syndrome in CIA rats. After successful modeling, the groups were randomly grouped, and the administered groups were gavaged with GE (60, 120 mg · kg

Effects of 2-dodecyl-6-methoxycyclohexa-2,5-diene-l ,4-dione on resisting hepatic fibrosis induced by CC14 in rats and its mechanisms via TGF-pi/Smads signaling pathway / 中国药理学通报

Aim To investigate the effects of 2-dode-cyl-6-methoxycyclohexa-2 , 5-diene-l, 4-dione ( DM-DD) on resisting hepatic fibrosis induced by carbon tetrachloride ( CC14 ) in rats and the underlying mechanisms , with a specific focus on the TGF-pi/Smads signaling pathway. Methods The hepatic fibrosis model was replicated using 50% CC14. Various parameters, including levels of aspartate transferase ( AST) , ala-nine transferase ( ALT ) , albumin/globulin ( A/G ) , total protein (TP) , total bilirubin (T-BIL) , hyaluron-ic acid ( HA ) , laminin ( LN ) , collagen type Ж ( Col Ж) , and collagen type IV(ColIV) in the blood, were measured. Liver tissue lesions and fiber formation were observed using HE and Masson staining. The expression levels of a smooth muscle actin (a-SMA) , collagen type I ( Col I ) , transformed growth factor (TGF-pi), Smad2, and Smad7 proteins were assessed using immunohistochemistry. a-SMA, Coll, TGF-pi, and Smad7 mRNA levels in liver tissue were measured by RT-PCR. Additionally, the expression levels of TGF-pi, Smad4, and Smad7 proteins in liver tissue were determined by Western blot. Results In comparison to the normal control group, the model group exhibited significantly elevated levels of AST, ALT, TP, T-BIL, HA, LN, Col Ш and Col IV in serum. But A/G level notably decreased. Successful modeling was confirmed by the presence of extensive fiber formations observed through HE and Massonstaining in liver tissue. The DMDD administration group demonstrated a notable decrease levels of AST, ALT, TP, T-BIL, HA, LN, Col III, and CollV, but A/G was significantly elevated when compared to the model group. Furthermore, a-SMA, Coll, TGF-f31, Smad2 and Smad4 mRNA and protein levels in the DMDD administration group were significantly reduced, while Smad7 significantly declined. HE and Masson staining results reflected a marked reduction in fibrous hyper-plasia. Conclusion DMDD exhibits a protective effect against CCl4-induced hepatic fibrosis, and its mechanism appears to be associated with the TGF-fJl/ Smads signaling pathway.

Effect of enriched environment combined with acupuncture at head points on behavior in rats with autism spectrum disorder / 中国康复理论与实践

ObjectiveTo observe the effect of enriched environment (EE) combined with acupuncture at head point (HA) on behavior in rats with autism spectrum disorder. MethodsHealthy female Wistar rats were given peritoneal injection of sodium valproate at 12.5 days of gestation. Twenty-four male offspring rats were randomly selected and then randomly divided into model group (n = 6), EE group (n = 6), HA group (n = 6) and EE combined with HA group (the combined group, n = 6). Six male offspring rats born from female mice injected with the same amount of saline intraperitoneally were as control group. After four weeks of treatment, all the five groups were tested with three-chamber test and marble burying test, and the sociability index, the social novelty index and the number of buried marbles were recorded. The levels of interleukin (IL)-1β and IL-6 in peripheral blood were determined by enzyme-linked immunosorbent assay (ELISA). ResultsAfter treatment, compared with the model group, the sociability index and the social novelty index improved (P < 0.05), the number of buried marbles reduced (P < 0.05), and the levels of IL-6 and IL-1β in peripheral blood decreased in EE group, HA group and the combined group (P < 0.05); while the combined group was the best (P < 0.01). ConclusionBoth EE or acupuncture at HA could improve behavioral symptoms, and reduce the expression of inflammatory factors in rats with autism spectrum disorder. The combination of the two methods showed the best result.

Protective mechanism of rhubarb decoction against inflammatory damage of brain tissue in rats with mild hepatic encephalopathy： A study based on the PI3K/AKT/mTOR signaling pathway / 临床肝胆病杂志

ObjectiveTo investigate the role and possible mechanism of action of rhubarb decoction （RD） retention enema in improving inflammatory damage of brain tissue in a rat model of mild hepatic encephalopathy （MHE）. MethodsA total of 60 male Sprague-Dawley rats were divided into blank group （CON group with 6 rats） and chronic liver cirrhosis modeling group with 54 rats using the complete randomization method. After 12 weeks， 40 rats with successful modeling which were confirmed to meet the requirements for MHE model by the Morris water maze test were randomly divided into model group （MOD group）， lactulose group （LT group）， low-dose RD group （RD1 group）， middle-dose RD group （RD2 group）， and high-dose RD group （RD3 group）， with 8 rats in each group. The rats in the CON group and the MOD group were given retention enema with 2 mL of normal saline once a day； the rats in the LT group were given retention enema with 2 mL of lactulose at a dose of 22.5% once a day； the rats in the RD1， RD2， and RD3 groups were given retention enema with 2 mL RD at a dose of 2.5， 5.0， and 7.5 g/kg， respectively， once a day. After 10 days of treatment， the Morris water maze test was performed to analyze the spatial learning and memory abilities of rats. The rats were analyzed from the following aspects： behavioral status； the serum levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） and the level of blood ammonia； pathological changes of liver tissue and brain tissue； the mRNA and protein expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （AKT）， and mammalian target of rapamycin （mTOR） in brain tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the MOD group， the RD1， RD2， and RD3 groups had a significantly shorter escape latency （all P<0.01）， significant reductions in the levels of ALT， AST， IL-1β， IL-6， TNF-α， and blood ammonia （all P<0.05）， significant alleviation of the degeneration， necrosis， and inflammation of hepatocytes and brain cells， and significant reductions in the mRNA and protein expression levels of PI3K， AKT， and mTOR in brain tissue （all P<0.05）， and the RD3 group had a better treatment outcome than the RD1 and RD2 groups. ConclusionRetention enema with RD can improve cognitive function and inflammatory damage of brain tissue in MHE rats， possibly by regulating the PI3K/AKT/mTOR signaling pathway.

Therapeutic effect of transplantation of bone marrow mesenchymal stem cells co-cultured with bone marrow M2 macrophages on a rat model of liver cirrhosis / 临床肝胆病杂志

ObjectiveTo investigate the effect of transplantation of bone marrow mesenchymal stem cells （BMSCs） co-cultured with bone marrow-derived M2 macrophages （M2-BMDMs）， named as BMSCM2， on a rat model of liver cirrhosis induced by carbon tetrachloride （CCl4）/2-acetaminofluorene （2-AAF）. MethodsRat BMDMs were isolated and polarized into M2 phenotype， and rat BMSCs were isolated and co-cultured with M2-BMDMs at the third generation to obtain BMSCM2. The rats were given subcutaneous injection of CCl4 for 6 weeks to establish a model of liver cirrhosis， and then they were randomly divided into model group （M group）， BMSC group， and BMSCM2 group， with 6 rats in each group. A normal group （N group） with 6 rats was also established. Since week 7， the model rats were given 2-AAF by gavage in addition to the subcutaneous injection of CCl4. Samples were collected at the end of week 10 to observe liver function， liver histopathology， and hydroxyproline （Hyp） content in liver tissue， as well as changes in the markers for hepatic stellate cells， hepatic progenitor cells， cholangiocytes， and hepatocytes. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the N group， the M group had significant increases in the activities of serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） （P<0.01）； compared with the M group， the BMSC and BMSCM2 groups had significant reductions in ALT and AST （P<0.01）， and the BMSCM2 group had significantly better activities than the BMSC group （P<0.05）. Compared with the N group， the M group had significant increases in Hyp content and the mRNA and protein expression levels of alpha-smooth muscle actin （α-SMA） in the liver （P<0.01）； compared with the M group， the BMSC and BMSCM2 groups had significant reductions in Hyp content and the expression of α-SMA （P<0.05）， and the BMSCM2 group had a significantly lower level of α-SMA than the BMSC group （P<0.01）. Compared with the N group， the M group had significant increases in the mRNA expression levels of the hepatic progenitor cell markers EpCam and Sox9 and the cholangiocyte markers CK7 and CK19 （P<0.01） and significant reductions in the expression levels of the hepatocyte markers HNF-4α and Alb （P<0.01）； compared with the M group， the BMSC and BMSCM2 groups had significant reductions in the mRNA expression levels of EpCam， Sox9， CK7， and CK19 （P<0.05） and significant increases in the mRNA expression levels of HNF-4α and Alb （P<0.05）， and compared with the BMSC group， the BMSCM2 group had significant reductions in the mRNA expression levels of EpCam and CK19 （P<0.05） and significant increase in the expression level of HNF-4α （P<0.05）. ConclusionM2-BMDMs can enhance the therapeutic effect of BMSCs on CCl4/2-AAF-induced liver cirrhosis in rats， which provides new ideas for further improving the therapeutic effect of BMSCs on liver cirrhosis.

Changes in the total volume and contour density of oval cells in hepatic lobules of rats with carbon tetrachloride-induced hepatic fibrosis： A stereological study / 临床肝胆病杂志

ObjectiveTo quantitatively investigate the changes in the total volume and contour density of hepatic oval cells （HOC） in hepatic lobules of rats with carbon tetrachloride （CCl4）-induced hepatic fibrosis. MethodsA total of 11 healthy male Sprague-Dawley rats were randomly divided into control group with 5 rats and hepatic fibrosis group with 6 rats， and CCl4 and olive oil suspension were injected subcutaneously twice a week， 3 mL/kg each time. After five weeks of hepatic fibrosis modeling， five liver tissue blocks with a size of about 1 mm3 were randomly selected from the liver of each rat to prepare one Epon812 epoxy resin-embedded ultrathin section， and the stereological method and transmission electron microscopy were used for the quantitative analysis of the total volume and contour density of HOC in the hepatic lobules of rats. In addition， four liver tissue blocks with a thickness of 2 mm were randomly selected from the remaining liver of each rat to prepare two paraffin-embedded Masson staining sections， and the degree of liver fibrosis in each rat was qualitatively evaluated according to the Metavir staging criteria for liver fibrosis. The independent-samples t test was used for comparison of continuous data between groups. ResultsThe quantitative stereological analysis showed that the total volume of HOC in hepatic lobules was 15.40±7.63 mm3 in the control group and 146.80±114.00 mm3 in the liver fibrosis group， and compared with the control group， the total volume of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 8.53 times （t=-2.551， P=0.031）； the contour density of HOC in hepatic lobules was 56.20±40.40 in the control group and 566.50±317.00 in the liver fibrosis group， and compared with the control group， the contour density of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 9.08 times （t=-3.539， P=0.006）. Qualitative observation showed that liver fibrosis stage of rats reached stage Ⅱ-Ⅲ according to the Metavir scoring criteria， and massive proliferation of HOC was observed around the proliferation site of hepatic stellate cells in the perisinusoidal space of rats. ConclusionCCl4 induces significant proliferation of HOC in hepatic lobules of rats with liver fibrosis.

Mechanism of Youguiwan Regulating BMP-2/Smad Signaling Pathway to Promote Bone Formation in Postmenopausal Rats with Osteoporosis / 中国实验方剂学杂志

ObjectiveTo observe the effects of Youguiwan on bone metabolism and bone morphogenetic protein-2 （BMP-2）/Smad signaling pathway in ovaries-removed rats with osteoporosis and study the mechanism of Youguiwan in the prevention and treatment of osteoporosis. MethodA postmenopausal rat model of osteoporosis was prepared by bilateral ovariectomy. The 40 female SD rats were randomly divided into five groups， including sham operation group， model group， alendronate sodium group （0.1 mg·kg-1）， and high-dose and low-dose （5.36 and 2.68 g·kg-1） groups of Youguiwan. The drug was given seven days after modeling， once a day for 12 weeks. After the treatment， the changes in femur tissue structure were observed by micro-CT， including bone mineral density （BMD）， bone volume/total volume （BV/TV）， trabecular number （Tb.N）， trabecular thickness （Tb.Th）， bone surface/bone volume （BS/BV）， and trabecular separation （Tb.Sp）. Ossification was observed by saffrane-solid green staining， and serum levels of bone metabolism markers， including bone alkaline phosphatase （BALP）， osteocalcin （BGP）， type Ⅰ procollagen amino terminal propeptide （PINP）， and tartrate-resistant acid phosphatase 5b （TRACP-5b）， were determined by enzyme-linked immunosorbent assay （ELISA）. The protein and mRNA expression levels of Runx2， BMP-2， and Smad1 in rat femur were detected by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham operation group， bone trabecula in the model group was sparse. BMD， BV/TV， Tb.N， and Tb.Th were decreased （P<0.05， P<0.01）. BS/BV （P<0.05） and Tb.Sp were increased. The content of BGP， BALP， PINP， and TRACP-5b in serum was significantly increased （P<0.01）. The mRNA and protein expressions of Runx2， BMP-2， and Smad1 in rat femur were significantly decreased （P<0.05， P<0.01）. Compared with the model group， the number of bone trabeculae in the high-dose and low-dose groups of Youguiwan was increased， and the bone microstructure was improved. BMD， BV/TV， Tb.N， and Tb.Th were increased significantly （P<0.05， P<0.01）， and BS/BV and Tb.Sp were increased. The content of bone metabolic markers decreased （P<0.05， P<0.01）. ConclusionYouguiwan has certain preventive and therapeutic effects on postmenopausal osteoporosis， and its mechanism may be related to promoting bone formation by regulating the BMP-2/Smad signaling pathway.

Earthworm Protein Protects Vascular Endothelial Function in Spontaneously Hypertensive Rats via PI3K/Akt/Nrf2 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of earthworm protein on the expression of phosphatidylinositol 3-kinase/protein kinase B/nuclear factor E2-related factor 2 （PI3K/Akt/Nrf2） pathway in the aorta of spontaneously hypertensive rats （SHR） and explore mechanism of earthworm protein in treating hypertensive vascular endothelial dysfunction （VED）. MethodTen 10-week-old Wistar Kyoto （WKY） rats and fifty SHR rats were selected for a week of adaptive feeding. WKY rats were selected as the normal group， and fifty SHR rats were randomized according to body weight into model， valsartan （8×10-3 g·kg-1·d-1）， and high-， medium-， and low-dose （0.2， 0.1， 0.05 g·kg-1·d-1， respectively） earthworm protein groups. The normal and model groups were administrated with equal volume of double distilled water by gavage. During the drug intervention period， the general situations of rats in each group were observed and their blood pressure was monitored at specific time points every other week before and after administration. After 8 weeks of drug intervention， enzyme-linked immunosorbent assay was employed to measure the levels of angiotensin-Ⅱ （Ang-Ⅱ） and endothelin-1 （ET-1） in the serum of rats in each group. The corresponding kits were used to determine the levels of nitric oxide （NO）， malondialdehyde （MDA）， glutathione peroxidase （GPX）， superoxide dismutase （SOD）， and ferrous ion （Fe2+）. Hematoxylin-eosin （HE） staining was employed to observe the changes in the intima of the aorta. Fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA levels of PI3K， Akt， Nrf2， heme oxygenase-1 （HO-1）， and glutathione peroxidase 4 （GPX4） in the aortic tissue. Western blotting was used to determine the protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the thoracic aorta. ResultCompared with the normal group， the model group had decreased body mass， increased irritability， severe endothelial damage， elevated blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， lowered NO level （P<0.01）， and down-regulated mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the aortic tissue （P<0.01）. Compared with the model group， drug intervention caused no significant change in the body mass， calmed the rats， alleviated the endothelial damage， lowered blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， elevated the NO level （P<0.05）， and up-regulated the mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 （P<0.05）. ConclusionThe earthworm protein can exert antihypertensive effects by ameliorating VED in SHR. Specifically， it may regulate the PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress and ferroptosis.

Oral glutamine dipeptide or oral glutamine free amino acid reduces burned injury progression in rats

Abstract This study was carried out to evaluate the effect of Glutamine, as a dipeptide or a free amino acid form, on the progression of burn injuries in rats. Thirty male Wistar rats were burned with a comb metal plate heated in boiling water (98 °C) for three minutes, creating four rectangular full-thickness burn areas separated by three unburned interspaces (zone of stasis) in both dorsum sides. The animals were randomized into three groups (n=10): saline solution (G1-Control) and treated groups that orally received Glutamine as dipeptide (G2-Dip) or free amino acid (G3-FreeAA). Two and seven days after burn injury, lesions were photographed for unburned interspaces necrosis evolution assessment. Seven days after injury, glutathione seric was measured and histopathological analysis was performed. By photographs, there was a significant reduction in necrosis progression in G3-Free-AA between days two and seven. Histopathological analysis at day 7 showed a significantly higher stasis zone without necrosis and a higher number of fibroblasts in G2-Dip and G3-FreeAA compared with G1-Control. Also, glutathione serum dosage was higher in G2-Dip. The plasmatic glutathione levels were higher in the G2-Dip than the G1-Control, and there was a trend to higher levels in G3-FreeAA. The reduction in histological lesions, greater production of fibroblasts, and greater amounts of glutathione may have benefited the evolution of burn necrosis, which showed greater preservation of interspaces.

Resumo Este estudo foi realizado para avaliar o efeito da Glutamina, como um dipeptídeo ou forma de aminoácido livre, na progressão de queimaduras em ratos. Trinta ratos Wistar machos foram queimados com um pente de metal aquecido em água fervente (98 °C) por três minutos, criando quatro áreas retangulares queimadas separadas por três interesespaços não queimados (zona de estase) em ambos os lados do dorso. Os animais foram randomizados em três grupos (n = 10): solução salina (G1-Controle) e grupos tratados que receberam glutamina via oral como dipeptídeo (G2-Dip) ou aminoácido livre (G3-FreeAA). Dois e sete dias após a queimadura, as lesões foram fotografadas para avaliação da evolução da necrose entre os espaços não queimados. Sete dias após a lesão, foi dosada a glutationa sérica e realizada análise histopatológica. Pelas fotografias, houve uma redução significativa na progressão da necrose no G3-Free-AA entre os dias dois e sete. A análise histopatológica no dia 7 mostrou uma zona de estase significativamente maior sem necrose e número mais elevado de fibroblastos em G2-Dip e G3-FreeAA em comparação com G1-Controle. Os níveis plasmáticos de glutationa foram maiores no G2-Dip em relação ao G1-Controle, e houve tendência a níveis mais elevados no G3-FreeAA. A redução das lesões histológicas, maior produção de fibroblastos, maior quantidade de glutationa podem ter beneficiado a evolução da necrose da queimadura, que mostrou maior preservação dos interespaços.

Effects of systemic ozone administration on the fresh extraction sockets healing: a histomorphometric and immunohistochemical study in rats

Abstract Studies have highlighted numerous benefits of ozone therapy in the field of medicine and dentistry, including its antimicrobial efficacy against various pathogenic microorganisms, its ability to modulate the immune system effectively, reduce inflammation, prevent hypoxia, and support tissue regeneration. However, its effects on dental extraction healing remain to be elucidated. Objective Therefore, this study aimed to evaluate the effects of systemically administered ozone (O3) at different doses in the healing of dental extraction sockets in rats. Methodology To this end, 72 Wistar rats were randomly divided into four groups after extraction of the right upper central incisor: Group C - control, no systemic treatment; Group OZ0.3 - animals received a single dose of 0.3 mg/kg O3; Group OZ0.7 - a single dose of 0.7 mg/kg O3; and Group OZ1.0 - a single dose of 1.0 mg/kg O3, intraperitoneally. In total, six animals from each group were euthanized at 7, 14, and 21 days after the commencement of treatment. Bone samples were harvested and further analyzed by descriptive histology, histomorphometry, and immunohistochemistry for osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) protein expression. Results All applied doses of O3 were shown to increase the percentage of bone tissue (PBT) after 21 days compared to group C. After 14 days, the OZ0.7 and OZ1.0 groups showed significantly higher PBT when compared to group C. The OZ1.0 group presented the most beneficial results regarding PBT among groups, which denotes a dose-dependent response. OCN immunostaining was higher in all groups at 21 days. However, after seven and 14 days, the OZ1.0 group showed a significant increase in OCN immunostaining compared to C group. No differences in TRAP+ osteoclasts were found between groups and time points. Conclusion Therefore, O3 therapy at higher doses might be beneficial for bone repair of the alveolar socket following tooth extraction.

A rapid and sensitive High-Performance Liquid Chromatography method with fluorescence detection for quantification of melatonin in small volume rat plasma samples: application to a preclinical study to determine the oral pharmacokinetics of melatonin under gestational conditions

Abstract A novel, simple and sensitive high-performance liquid chromatography with fluorescence detection method was developed and validated for the characterization of the preclinical pharmacokinetics of melatonin under pregnant conditions. Plasma samples (25 µL) were treated with 30 µL of ethanol absolute (containing the internal standard, IS). After a centrifugation process, aliquots of supernant (5 µL) were injected into the chromatographic system. Compounds were eluted on a Xbridge C18 (150 mm x 4.6 mm i.d., 5 µm particle size) maintained at 30°C. The mobile phase consisted in a mixture of aqueous solution of 0.4% phosphoric acid and acetonitrile (70:30 v/v). The wavelengths were set at 305 nm (excitation) and 408 nm (emission) and the total analysis time was 8 min/sample. All validation tests were obtained with accuracy and precision, according to FDA guidelines, over the concentration range of 0.005-20 µg/mL. Pharmacokinetic study showed that melatonin systemic exposure increased from day 14, with a significant difference at 19 days of gestation compared to the control group. Our findings suggest a decreased metabolism of melatonin as result of temporary physiological changes that occur throughout pregnancy. However, other maternal physiological changes cannot be ruled out.

Methylene blue as an adjuvant during cardiopulmonary resuscitation: an experimental study in rats

Abstract Introduction: Methylene Blue (MB) has been shown to attenuate oxidative, inflammatory, myocardial, and neurological lesions during ischemia-reperfusion and has great potential during cardiac arrest. This study aimed to determine the effects of MB combined with epinephrine during cardiac arrest on myocardial and cerebral lesions. Method: Thirty-eight male Wistar rats were randomly assigned to four groups: the sham group (SH, n = 5), and three groups subjected to cardiac arrest (n = 11 /group) and treated with EPI 20 μg.kg−1 (EPI), EPI 20 μg.kg−1 + MB 2 mg.kg−1 (EPI + MB), or saline 0.9% 0.2 ml (CTL). Ventricular fibrillation was induced by direct electrical stimulation in the right ventricle for 3 minutes, and anoxia was maintained for 5 minutes. Cardiopulmonary Resuscitation (CPR) consisted of medications, ventilation, chest compressions, and defibrillation. After returning to spontaneous circulation, animals were observed for four hours. Blood gas, troponin, oxidative stress, histology, and TUNEL staining measurements were analyzed. Groups were compared using generalized estimating equations. Results: No differences in the Returning of Spontaneous Circulation (ROSC) rate were observed among the groups (EPI: 63%, EPI + MB: 45%, CTL: 40%, p = 0.672). The mean arterial pressure immediately after ROSC was higher in the EPI+MB group than in the CTRL group (CTL: 30.5 [5.8], EPI: 63 [25.5], EPI+MB: 123 [31] mmHg, p = 0.007). Serum troponin levels were high in the CTL group (CTL: 130.1 [333.8], EPI: 3.70 [36.0], EPI +MB: 43.7 [116.31] ng/mL, p < 0.05). Conclusion: The coadministration of MB and epinephrine failed to yield enhancements in cardiac or brain lesions in a rodent model of cardiac arrest.

Oral glutamine dipeptide or oral glutamine free amino acid reduces burned injury progression in rats / O dipeptídeo de glutamina oral ou aminoácido livre de glutamina oral reduz a progressão da lesão por queimadura em ratos

Abstract This study was carried out to evaluate the effect of Glutamine, as a dipeptide or a free amino acid form, on the progression of burn injuries in rats. Thirty male Wistar rats were burned with a comb metal plate heated in boiling water (98 °C) for three minutes, creating four rectangular full-thickness burn areas separated by three unburned interspaces (zone of stasis) in both dorsum sides. The animals were randomized into three groups (n=10): saline solution (G1-Control) and treated groups that orally received Glutamine as dipeptide (G2-Dip) or free amino acid (G3-FreeAA). Two and seven days after burn injury, lesions were photographed for unburned interspaces necrosis evolution assessment. Seven days after injury, glutathione seric was measured and histopathological analysis was performed. By photographs, there was a significant reduction in necrosis progression in G3-Free-AA between days two and seven. Histopathological analysis at day 7 showed a significantly higher stasis zone without necrosis and a higher number of fibroblasts in G2-Dip and G3-FreeAA compared with G1-Control. Also, glutathione serum dosage was higher in G2-Dip. The plasmatic glutathione levels were higher in the G2-Dip than the G1-Control, and there was a trend to higher levels in G3-FreeAA. The reduction in histological lesions, greater production of fibroblasts, and greater amounts of glutathione may have benefited the evolution of burn necrosis, which showed greater preservation of interspaces.

Resumo Este estudo foi realizado para avaliar o efeito da Glutamina, como um dipeptídeo ou forma de aminoácido livre, na progressão de queimaduras em ratos. Trinta ratos Wistar machos foram queimados com um pente de metal aquecido em água fervente (98 °C) por três minutos, criando quatro áreas retangulares queimadas separadas por três interesespaços não queimados (zona de estase) em ambos os lados do dorso. Os animais foram randomizados em três grupos (n = 10): solução salina (G1-Controle) e grupos tratados que receberam glutamina via oral como dipeptídeo (G2-Dip) ou aminoácido livre (G3-FreeAA). Dois e sete dias após a queimadura, as lesões foram fotografadas para avaliação da evolução da necrose entre os espaços não queimados. Sete dias após a lesão, foi dosada a glutationa sérica e realizada análise histopatológica. Pelas fotografias, houve uma redução significativa na progressão da necrose no G3-Free-AA entre os dias dois e sete. A análise histopatológica no dia 7 mostrou uma zona de estase significativamente maior sem necrose e número mais elevado de fibroblastos em G2-Dip e G3-FreeAA em comparação com G1-Controle. Os níveis plasmáticos de glutationa foram maiores no G2-Dip em relação ao G1-Controle, e houve tendência a níveis mais elevados no G3-FreeAA. A redução das lesões histológicas, maior produção de fibroblastos, maior quantidade de glutationa podem ter beneficiado a evolução da necrose da queimadura, que mostrou maior preservação dos interespaços.

Cilostazol protects against changes caused by streptozotocin-induced diabetic retinopathy / Cilostazol protege contra as alterações causadas pela retinopatia diabética induzida por estreptozotocina

ABSTRACT Purpose: This study investigates the protective effect of cilostazol on the development and evolution of diabetic retinopathy in rats. Methods: Sixty male rats were divided into four groups: untreated nondiabetic rats, untreated diabetic rats, cilostazol-treated nondiabetic rats, and cilostazol-treated diabetic rats. The thickness of the internal limiting membrane to the outer limiting membrane, inner plexiform layer, inner nuclear layer, and outer nuclear layer were measured. The number of cell nuclei per 50-μm length in retinal sections was counted to quantify the degree of retinal cell loss. Results: The number of nuclei in the ganglion cell layer was significantly higher in untreated nondiabetic rats (p<0.05). The mean number of nuclei in the cilostazol-treated nondiabetic rats was significantly higher than that in the cilostazol-treated diabetic rats (p<0.05). The cilostazol-treated nondiabetic rats had a significantly higher mean nuclei count in the inner nuclear layer and inner plexiform layer as compared with the other groups (p<0.05). The total mean retinal thickness of the cilostazol-treated nondiabetic rats was significantly higher than that of cilostazol-treated diabetic rats and untreated diabetic rats (p<0.05). Conclusion: By decreasing the loss of ganglion cells and reducing the sensorineural atrophy in the internal retinal layers, cilostazol had a protective effect against changes caused by diabetic retinopathy in diabetic rats.

RESUMO Objetivo: O objetivo deste estudo foi investigar o efeito protetor do cilostazol no desenvolvimento e na evolução da retinopatia diabética em ratos. Métodos: Sessenta ratos machos foram divididos em 4 grupos: ratos não-diabéticos não-tratados, ratos diabéticos não-tratados, ratos não-diabéticos tratados com cilostazol e ratos diabéticos tratados com cilostazol. A espessura da membrana limitante interna à membrana limitante externa, a camada plexiforme interna, a camada nuclear interna e a camada nuclear externa foram medidas. Para quantificar o grau de perda de células da retina, foi contado o número de núcleos de células por 50 μm de comprimento em secções retinianas. Resultados: O número de núcleos no GCL foi significativamente maior em Ratos não-diabéticos não-tratados com cilostazol (p<0,05). O número médio de núcleos em Ratos não-diabéticos tratados com cilostazol foi significativamente maior do que em Ratos diabéticos tratados com cilostazol (p<0,05). A contagem média de núcleos em camada nuclear interna e camada plexiforme interna de ratos não-diabéticos tratados com cilostazol foi significativamente maior do que nos outros grupos (p<0,05). A espessura retiniana média total de Ratos não-diabéticos tratados com cilostazol foi significativamente maior do que em Ratos diabéticos tratados com cilostazol e Ratos diabéticos não-tratados (p<0,05). Conclusão: Os resultados demonstraram que o cilostazol teve um efeito protetor contra as alterações causadas pela retinopatia diabética em ratos diabéticos, diminuindo a perda de células ganglionares e reduzindo a atrofia neurossensorial nas camadas retinianas internas.

Garlic Extract Reverses Ischemic Heart Disease in Female Rats Exposed to Combined Oral Contraceptive Pills

Abstract Background Garlic is an herbal medicinal plant with several pharmacological properties used for the management of various ailments. However, its potential in the reversal of ischemic heart disease induced by combined oral contraceptive pills is not well reported. Objective This study investigated the cardioprotective potential of aqueous garlic extract against cardiotoxicity induced by oral contraceptive pills. Methods Forty-six rats were used for this study. Six were used to determine the lethal dose (LD50), and 40 rats were divided randomly into 5 groups of 8 rats each. Group A received feed and distilled water. Group B received 0.6 mg/kg of oral contraceptive pills. Group C received 500 mg/kg of garlic extract. Group D received 0.6 mg/kg of oral contraceptive pills + 500 mg/kg garlic extract. Group E received 0.6 mg/kg of oral contraceptive pills + 700 mg/kg garlic extract. The animals were sacrificed, and blood and tissue samples were collected for biochemical and histological analysis. Statistical analysis was done using SPSS, and p values < 0.05 were considered significant. Results The acute toxicity dose of combined oral contraceptive pills was 1.5 mg/kg for albino rats. Combined oral contraceptive pills induced ischemic necrosis as revealed by the photomicrographs, in addition to elevation of serum cardiac troponin-1, lactate dehydrogenase, creatine kinase, and malondialdehyde levels. Treatment with garlic extract demonstrated significant reduction in cardiac troponin-1 (p = 0.000), lactate dehydrogenase (p = 0.002), creatine kinase (p = 0.001), and malondialdehyde (p = 0.001) levels, as well as restoration of the cardiac cytoarchitecture changes caused by the combined oral contraceptive pills. Conclusion This study has demonstrated that aqueous garlic juice can reverse ischemic heart disease, lessen cytoarchitectural alterations of the heart caused by combined oral contraceptive pills, and thus ameliorate cardiac dysfunction.

Preclinical study of experimental burns treated with photobiomodulation and Human Amniotic Membrane, both isolated and associated / Estudo pré-clínico de queimaduras experimentais tratadas com fotobiomodulação e membrana amniótica humana, isoladas e associadas / Estudio preclínico de quemaduras experimentales tratadas con fotobiomodulación y membrana amniótica humana, solas y combinadas

Abstract Objective: to evaluate the effect of photobiomodulation with low-level 660 nm laser alone or associated with Human Amniotic Membrane in the repair of partial-thickness burns in rats. Method: an experimental study conducted with 48 male Wistar rats, randomized into four groups: Control, Human Amniotic Membrane, Low-Level Laser Therapy, and Low-Level Laser Therapy associated with Human Amniotic Membrane. The histopathological characteristics of the skin samples were analyzed 7 and 14 days after the burn. The data obtained were submitted to the Kolmogorov-Smirnov and Mann-Whitney tests. Results: the histological analysis of the burn injuries showed a decrease in inflammation (p<0.0001) and an increase in proliferation of fibroblasts (p<0.0001) mainly at 7 days in all treatments related to the control group. At 14 days, the greater effectiveness in accelerating the healing process was significant (p<0.0001) in the Low-Level Laser Therapy group associated with the Human Amniotic Membrane. Conclusion: the association of photobiomodulation therapies with the Human Amniotic Membrane allowed verifying a reduction in the healing process time of the experimental lesions, stimulating its proposal as a treatment protocol in partial-thickness burns.

Resumo Objetivo: avaliar o efeito da fotobiomodulação com laser de baixa intensidade 660 nm isoladamente ou associada à membrana amniótica humana no reparo de queimaduras de espessura parcial em ratos. Método: estudo experimental com 48 ratos Wistar machos, randomizados em quatro grupos: Controle, Membrana Amniótica Humana, Terapia a Laser de Baixa Intensidade e Terapia a Laser de Baixa Intensidade associado à Membrana Amniótica Humana. As características histopatológicas das amostras de pele foram analisadas aos 7 e 14 dias após a queimadura. Os dados obtidos foram submetidos aos testes de Kolmogorov-Smirnov e Mann Whitney. Resultados: a análise histológica das lesões por queimadura mostrou a diminuição da inflamação (p <0,0001) e aumento da proliferação de fibroblastos (p <0,0001), principalmente nos 7 dias em todos os tratamentos relacionados ao grupo controle. Aos 14 dias, a maior efetividade na aceleração do processo cicatricial foi significativa (p<0,0001) no grupo Terapia a Laser de Baixa Intensidade associado à Membrana Amniótica Humana. Conclusão: a associação das terapias de fotobiomodulação à membrana amniótica humana permitiu comprovar redução no tempo do processo cicatricial das lesões experimentais, estimulando sua proposição como protocolo de tratamento em queimaduras de espessura parcial.

Resumen Objetivo: evaluar el efecto de la fotobiomodulación con láser de baja intensidad 660 nm de sola o combinada con la membrana amniótica humana en la reparación de quemaduras de espesor parcial en ratas. Método: estudio experimental con 48 ratas Wistar macho, aleatorizadas en cuatro grupos: Control, Membrana Amniótica Humana, Terapia con Láser de Baja Intensidad y Terapia con Láser de Baja Intensidad combinada con la Membrana Amniótica Humana. Las características histopatológicas de las muestras de piel fueron analizadas a los 7 y 14 días después de la quemadura. Los datos obtenidos fueron sometidos a las pruebas de Kolmogorov-Smirnov y Mann-Whitney. Resultados: el análisis histológico de las lesiones por quemadura mostró una disminución de la inflamación (p <0,0001) y un aumento de la proliferación de fibroblastos (p <0,0001) principalmente a los 7 días en todos los tratamientos en comparación con el grupo control; a los 14 días, en el grupo de Terapia con Láser de Baja Intensidad combinada con la Membrana Amniótica Humana la mayor efectividad en la aceleración del proceso de cicatrización fue significativa (p<0,0001). Conclusión: la asociación de terapias de fotobiomodulación con la membrana amniótica humana permitió comprobar que hubo una reducción en el tiempo del proceso de cicatrización de lesiones experimentales, lo cual favorece que se proponga como protocolo de tratamiento en quemaduras de espesor parcial.

Estudios de Seguridad Preclínica del extracto hidroalcohólico de semillas de Opuntia soehrensii, una especie alto-andina con actividad anti-Herpes Virus, en ratas Sprague Dawley / Preclinical safety of the hydroalcoholic extract of Opuntia soehrensii seeds with anti-herpes virus activity, in SD rats

Introducción. Alrededor de 3700 millones menores de 50 años con infección por VHS-1 y 491 millones de personas de 15 a 49 años cursan con infección por VHS-2 en el mundo; sus síntomas, vesículas o ulceras dolorosas reaparecen periódicamente. El tratamiento convencional disminuyó su efectividad en cepas resistentes e inmunodeprimidos. Alternativas terapéuticas con extractos de plantas medicinales y potencial antiviral, como Opuntia soehrensii Brito conocida como "ayrampù" en Bolivia, utiliza infusión de sus semillas como analgésico, antidiabético, hipotensor y febrífugo. En vapores por inhalación para afecciones respiratorias; como tintura tópica en lesiones dérmicas de viruela, sarampión y herpes labial. Objetivo. Evaluar la seguridad preclínica de un gel que contiene el extracto hidro-alcohólico de semillas de Opuntia soehrensii en diferentes dosis, aplicado en la mucosa vaginal de ratas Sprague Dawley. Material y métodos. Se ejecutaron protocolos de toxicidad aguda y subaguda para evaluar la respuesta sistémica, a través de marcadores bioquímicos y de comportamiento, y la respuesta local en mucosa vaginal, mediante estudios histopatológicos, en grupos de animales a los que se aplicó el gel con diferentes concentraciones del extracto de Opuntia soehrensii, comparados con un grupo control y otro que recibió solo el vehículo. Resultados. Se encontró que los indicadores sistémicos de comportamiento y ganancia de peso no mostraron diferencias entre grupos. Los indicadores hematológicos y bioquímicos mostraron resultados fisiológicamente esperados y sin cambios en los grupos de estudio. La citología expuso conservación del fenotipo celular para las fases del ciclo estral en todos los grupos. Los indicadores histológicos de reacción local e integridad celular se distribuyeron de igual manera en los todos los grupos. Conclusión. La aplicación de un gel de Opuntia soehrensii no muestra niveles apreciables de toxicidad local y sistémica, lo que permite recomendar la iniciación de estudios de aplicación clínica.

Introduction. Around 3.7 billion people under 50 years of age are infected with HSV-1 and 491 million people between the ages of 15 and 49 are infected with HSV-2 in the world; his symptoms, vesicles or painful ulcers recur periodically. Conventional treatment decreased its effectiveness in resistant and immunosuppressed strains. Therapeutic alternatives with extracts of medicinal plants and antiviral potential, such as Opuntia soehrensii Brito known as "ayrampù" in Bolivia, uses infusion of its seeds as an analgesic, antidiabetic, hypotensive and febrifuge. In vapors by inhalation for respiratory conditions; as a topical tincture in skin lesions of smallpox, measles and cold sores. Objectives . To evaluate the preclinical safety of a gel containing the hydroalcoholic extract of Opuntia soehrensii seeds in different doses, applied to the vaginal mucosa of Sprague Dawley rats. Material and Methods. Acute and sub-acute toxicity protocols were carried out to evaluate local response in the vaginal mucosa, through histo pathological studies, and systemic responses, through biochemical and behavioral markers, in groups of animals to which the gel with different concentrations of the extract of Opuntia soehrensii was applied, compared with a control group and another that received only the vehicle. Results. It was found that the histological indicators of local reaction and cell integrity were equally distributed in all groups. Cytology showed conservation of the cell phenotype for the phases of the estrous cycle in all groups. The systemic indicators of behavior and weight gain did not show differences between groups. Hematological and biochemical indicators showed results ranged in physiologic parameters, without changes in the study groups. Conclusion. The application of a gel from Opuntia soehrensii does not show appreciable levels of local and systemic toxicity, which makes it possible to recommend the initiation of clinical application studies.