Silencing ZKSCAN3 gene expression aggravates lung injury induced by LPS in mice / 中国临床药理学与治疗学

AIM: To explore the role of ZKSCAN3 in acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: After verifying the efficacy of ZKSCAN3 siRNA, male C57BL/6 mice were randomly divided into 4 groups (n = 8): control group(group A), LPS group (group B), scrambled siRNA group (group C) and ZKSCAN3 siRNA group (group D). Mice in groups A and B were given 1 mL of PBS via tail vein; mice in groups C were given corresponding doses of PBS containing scrambled siRNA; and mice in group D were given corresponding doses of RNase-free PBS containing ZKSCAN3 siRNA (50 μg). After 24 hours, mice in groups B, C, and D were instilled with LPS solution (5 mg/kg) through tracheal intubation to create an ALI model; group A was given the corresponding dose of PBS (20 ΜL). The samples were collected and tested 24 hours after the modeling administration. RESULTS: Compared with group B, silencing ZKSCAN3 gene expression reduced SOD activity and Bcl-2 level; while MDA, Bax and caspase-3 increased; correspondingly, the content of protein and cells in BAL, the apoptosis rate of lung tissue and the pathological score significantly increased (P < 0.05).CONCLUSION: Silencing ZKSCAN3 gene expression aggravates the lung injury caused by LPS, which may aggravate the pathological damage of lung tissue in mice by weakening the antioxidant function and aggravating tissue necrosis.

EPCs-exos combined with tanshinone Ⅱ_A protect vascular endothelium cells from oxidative damage via PI3K/Akt pathway / 中国中药杂志

This study aims to investigate the molecular mechanism of tanshinone Ⅱ_(A )(TaⅡ_A) combined with endothelial progenitor cells-derived exosomes(EPCs-exos) in protecting the aortic vascular endothelial cells(AVECs) from oxidative damage via the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt) pathway. The AVECs induced by 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine(POVPC) were randomly divided into model, TaⅡ_A, EPCs-exos, and TaⅡ_A+EPCs-exos groups, and the normal cells were taken as the control group. The cell counting kit-8(CCK-8) was used to examine the cell proliferation. The lactate dehydrogenase(LDH) cytotoxicity assay kit, Matrigel assay, DCFH-DA fluorescent probe, and laser confocal microscopy were employed to examine the LDH release, tube-forming ability, cellular reactive oxygen species(ROS) level, and endothelial cell skeleton morphology, respectively. The enzyme-linked immunosorbent assay was employed to measure the expression of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to determine the mRNA and protein levels, respectively, of PI3K and Akt. Compared with the control group, the model group showed decreased cell proliferation and tube-forming ability, increased LDH release, elevated ROS level, obvious cytoskeletal disruption, increased expression of IL-1β, IL-6, and TNF-α, and down-regulated mRNA and protein levels of PI3K and Akt. Compared with the model group, TaⅡ_A or EPCs-exos alone increased the cell proliferation and tube-forming ability, reduced LDH release, lowered the ROS level, repaired the damaged skeleton, decreased the expression of IL-1β, IL-6, and TNF-α, and up-regulated the mRNA and protein levels of PI3K and Akt. TaⅡ_A+EPCs-exos outperformed TaⅡ_A or EPCs-exos alone in regulating the above indexes. The results demonstrated that TaⅡ_A and EPCs-exos exerted a protective effect on POVPC-induced AVECs by activating the PI3K/Akt pathway, and the combination of the two had stronger therapeutic effect.

Mechanism of astragaloside Ⅳ in regulating autophagy of PC12 cells under oxygen-glucose deprivation by medicating Akt/mTOR/HIF-1α pathway / 中国中药杂志

This study explored the protective effect of astragaloside Ⅳ(AS-Ⅳ) on oxygen-glucose deprivation(OGD)-induced autophagic injury in PC12 cells and its underlying mechanism. An OGD-induced autophagic injury model in vitro was established in PC12 cells. The cells were divided into a normal group, an OGD group, low-, medium-, and high-dose AS-Ⅳ groups, and a positive drug dexmedetomidine(DEX) group. Cell viability was measured using the MTT assay. Transmission electron microscopy was used to observe autophagosomes and autolysosomes, and the MDC staining method was used to assess the fluorescence intensity of autophagosomes. Western blot was conducted to determine the relative expression levels of functional proteins LC3-Ⅱ/LC3-Ⅰ, Beclin1, p-Akt/Akt, p-mTOR/mTOR, and HIF-1α. Compared with the normal group, the OGD group exhibited a significant decrease in cell viability(P<0.01), an increase in autophagosomes(P<0.01), enhanced fluorescence intensity of autophagosomes(P<0.01), up-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and down-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.05 or P<0.01). Compared with the OGD group, the low-and medium-dose AS-Ⅳ groups and the DEX group showed a significant increase in cell viability(P<0.01), decreased autophagosomes(P<0.01), weakened fluorescence intensity of autophagosomes(P<0.01), down-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and up-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.01). AS-Ⅳ at low and medium doses exerted a protective effect against OGD-induced autophagic injury in PC12 cells by activating the Akt/mTOR pathway, subsequently influencing HIF-1α. The high-dose AS-Ⅳ group did not show a statistically significant difference compared with the OGD group. This study provides a certain target reference for the prevention and treatment of OGD-induced cellular autophagic injury by AS-Ⅳ and accumulates laboratory data for the secondary development of Astragali Radix and AS-Ⅳ.

Effect of Huatan Tongluo Decoction on Gut-Brain Axis in Rats with Cerebral Ischemia/Reperfusion / 中国实验方剂学杂志

Objective:To explore the effects of Huatan Tongluo Decoction （HTTLD） on the morphology and function of brain tissues and intestine in rats with cerebral ischemia/reperfusion based on the gut-brain axis. Method:Sixty SPF male rats were randomly divided into a sham operation group， a model group， high- （28.66 g·kg^-1）， medium- （14.33 g·kg^-1）， and low-dose （7.16 g·kg^-1） HTTLD groups， and an edaravone （4 g·kg^-1）+<italic>Clostridium butyricum</italic> （5.0×10⁸ cfu·mL^-1） group. The model was established by focal cerebral ischemia/reperfusion in rats. The drugs were administered by gavage. The brain tissue injury was determined by neurological deficit score and 2，3，5-triphenyl tetrazolium chloride （TTC） staining. The effect of cerebral ischemia/reperfusion on intestinal motility was assessed by the propulsion rate of small intestine. The intestinal mucosal cell damage was evaluated by the pathomorphological examination of the duodenal mucosa. Enzyme-linked immunosorbent assay （ELISA） was used to determine the content of <italic>D</italic>-lactate （<italic>D</italic>-LAC）， diamine oxidase （DAO）， and bacterial endotoxin （lipopolysaccharide， LPS） in serum. Western blot was used to detect the expression of Occludin， Claudin-5， and zonula occludens 1 （ZO-1） in the duodenum. Result:After cerebral ischemia/reperfusion， rats developed neurological deficit symptoms. The neurological deficit score in the model group was higher than that in the sham operation group （<italic>P<</italic>0.01）. Compared with the model group， the high- and medium-dose HTTLD groups could relieve the symptoms of neurological deficits and lower neurological deficit scores （<italic>P<</italic>0.01）. The results of TTC staining showed that the model group presented obvious infarcts in brain tissues compared with the sham operation group （<italic>P<</italic>0.01）. The cerebral infarction volumes of HTTLD groups were reduced compared with that in the model group （<italic>P<</italic>0.01）， especially the high-dose HTTLD group， and the effect was dose-dependent. Furthermore， the propulsion rate of small intestine in the model group was significantly reduced compared with that in the sham operation group （<italic>P<</italic>0.01）. Compared with the model group， HTTLD groups could increase propulsion rates of small intestine （<italic>P<</italic>0.01）， especially the high-dose HTTLD group， and the effect was dose-dependent. After cerebral ischemia/reperfusion， obvious duodenal mucosal damage could be observed， which was relieved after the administration of HTTLD. Western blot results showed that the protein expression of ZO-1， Occludin， and Claudin-5 in the model group was reduced compared with that in the sham operation group （<italic>P<</italic>0.01）. Compared with the model group， the HTTLD groups could up-regulate the expression of ZO-1， Occludin， and Claudin-5 to varying degrees （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， especially the high-dose HTTLD group. ELISA showed that the serum <italic>D</italic>-LAC， DAO， and LPS of the model group were elevated compared with those in the sham operation group （<italic>P<</italic>0.01）. Compared with the model group， the HTTLD groups showed reduced <italic>D</italic>-LAC and DAO （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， and the medium- and high-dose HTTLD groups showed reduced LPS （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， especially the high-dose HTTLD group. Conclusion:After cerebral ischemia/reperfusion， the rats showed damaged brain tissues， neurological dysfunction， intestinal mucosal injury， weakened intestinal motility， and destroyed the intestinal mucosal barrier. HTTLD can protect against brain-gut axis injury after cerebral ischemia/reperfusion by reducing the damage on brain tissues and gastrointestinal mucosa， relieving the symptoms of neurological deficits， promoting gastrointestinal motility， improving intestinal barrier function， and reducing the release of intestinal bacterial metabolites or poisons.

Effect of Astragaloside Ⅳ Combined with Notoginseng Total Saponins on Angiogenesis After Transplantation of BMSCs in Rats with Cerebral Ischemia / 中国实验方剂学杂志

Objective:To investigate the effect of astragaloside Ⅳ（AST Ⅳ）and Notoginseng total saponins （NTS） combined with bone marrow mesenchymal stem cell （BMSC） transplantation on neural repair and angiogenesis in rats with cerebral ischemia. Method:The rats were randomly divided into a sham operation group， a model group， low- and high-dose AST Ⅳ + NTS groups， a BMSC infusion group， and low- and high-dose BMSC infusion+AST Ⅳ （10 and 20 mg·kg^-1） + NTS group （25， 50 mg·kg^-1）. BMSCs were isolated and purified by whole bone marrow adherent culture. The positive expression of surface markers of BMSCs （CD29， CD90， CD34， and CD45） was detected by flow cytometry. The focal cerebral ischemia model was established by middle cerebral artery occlusion （MCAO）. The PKH26-labeled BMSCs were injected into the tail vein of rats in the BMSC infusion group， once a day. The rats in the combination groups received BMSC injection once a day and intragastric administration of drugs twice a day. Other groups were administered twice a day by gavage. The sham operation group and the model group received the same amount of normal saline. Symptoms and signs of neurological deficits were assessed by the Longa method and the cerebral infarction rate was determined by TTC staining. The survival and vascularization ［double positive expression of PKH26/vascular endothelial growth factor （VEGF）］ after transplantation of BMSCs were observed by the immunofluorescence method. The protein expression of Ang1 and TGF-<italic>β</italic>₁ was measured by Western blot. Result:BMSCs were properly isolated and cultured. The identification of surface markers CD29， CD90， CD34， and CD45 was consistent with the characteristics of BMSCs. The neurological deficit score and cerebral infarction rate of the model group were significantly increased （<italic>P</italic><0.01）. All drugs and cell transplantation could alleviate the above pathological changes in varying degrees. The strongest effect was observed in high-dose BMSC infusion+AST Ⅳ+NTS group （<italic>P</italic><0.01）， which was superior to those in the AST Ⅳ+NTS groups or the BMSC infusion group. BMSC injection helped cells survive in the ischemic brain tissues and promoted angiogenesis， and this effect could be enhanced by the combination with drugs. After cerebral ischemia， the expression of Ang1 and TGF-<italic>β</italic>₁ was increased， and the effect in the BMSC infusion+AST Ⅳ+NTS groups was the strongest （<italic>P</italic><0.01）. Conclusion:AST Ⅳ combined with NTS can promote the survival of transplanted BMSCs and facilitate angiogenesis after target repair of damaged blood vessels after cerebral ischemia. The mechanism may be related to the improvement of the local microenvironment in the brain after cerebral ischemia and the promotion of the survival and differentiation of transplanted stem cells.

On the effect of experimental animal ethics education on the formation of medical students' medical literacy / 中华医学教育探索杂志

Hunan University of Chinese Medicine has improved the implementation of animal ethics education from the aspects of carrying out elective courses, emphasizing the implementation of pre-class learning, integrating ethics education into experimental teaching, cultivating students' good medical literacy in a subtle way, and further deepening their learning by offering elective courses of animal ethics. The results show that the opening of elective courses and the enforcement of experimental animal ethics education in experimental classes have greatly strengthened the students' experimental animal ethics, which is conducive to the formation of students' medical literacy.

Role of PI3K∕Akt signaling pathway in propofol-induced reduction of intestinal ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective To evaluate the role of phosphatidylinositol 3-kinase ( PI3K)∕protein kinase B ( Akt) signaling pathway in propofol-induced reduction of intestinal ischemia-reperfusion ( I∕R) injury in rats. Methods Thirty-two healthy male Sprague-Dawley rats, aged 2-3 months, weighing 225-275 g, were divided into 4 groups ( n=8 each) using a random number table method: sham operation group ( Sham group), intestinal I∕R group ( I∕R group), propofol group ( P group), and PI3K inhibitor wortmannin plus propofol group ( W+P group) . Intestinal ischemia was induced by occluding the superior mesenteric ar-tery for 45 min followed by 2 h of reperfusion to establish the model of intestinal I∕R injury. Propofol was in-travenously infused at a rate of 20 mg·kg-1 ·h-1 starting from the onset of reperfusion until the end of reper-fusion in group P. Wortmannin 15 μg∕kg was intravenously injected at 25 min before reperfusion, and propofol was intravenously infused at a rate of 20 mg·kg-1 ·h-1 starting from the onset of reperfusion until the end of reperfusion in group W+P. Rats were sacrificed at 2 h of reperfusion, and small intestinal tissues were obtained for microscopic examination of pathologic changes of intestinal mucosa and for determination of wet∕dry weight ratio (W∕D ratio), malondialdehyde (MDA) content (by thiobarbituric acid colorimetric method) , superoxide dismutase ( SOD ) activity ( using xanthine oxidase method ) , myeloperoxidase ( MPO) activity ( by MPO assay) , and phosphorylated Akt ( p-Akt) expression ( by Western blot) . Intes-tinal damage was assessed and scored according to Chiu. Results Compared with group Sham, Chiu' s score, W∕D ratio, MDA content and MPO activity were significantly increased, the SOD activity was de-creased, and p-Akt expression was down-regulated in group I∕R (P<0. 05). Compared with group I∕R, Chiu's score, W∕D ratio, MDA content and MPO activity were significantly decreased, the SOD activity was increased, and p-Akt expression was up-regulated in group P (P<0. 05). Compared with group P, Chiu's score, W∕D ratio, MDA content and MPO activity were significantly increased, the SOD activity was decreased, and p-Akt expression was down-regulated in group W+P (P<0. 05). Conclusion The mechanism by which propofol reduces intestrnal I∕R injury is related to activating PI3K∕Akt signaling path-way and inhibiting inflammatory and oxidative stress responses in rats.

Effect of borneol combined with astragaloside IV and Panax notoginseng saponins on permeability of blood-brain barrier after cerebral ischemia-reperfusion / 中国药理学通报

Aim To probe the effect of borneol combined with astragalosides IV (AST I V) and Panax notoginseng saponins (PNS) on blood-brain barrier (BBB) after cerebral ischemia-reperfusion. Methods Rats with cerebral ischemia-reperfusion were administered by gavage, the symptom of nerve function defect was observed, the water content of brain tissues, the permeability of BBB, and the expression of zonula cccludens 1 (ZO-1), ZO-2, Occludin and Claudin-5 were detected. Results After cerebral ischemia-reperfusion, the neurological deficit symptom appeared, the neurological function score and brain water content increased, and BBB was destroyed. Each drug could relieve the above pathological changes to various degrees, and the effect of borneol + AST IV + PNS was the best, which was stronger than that of single drug and AST IV + PNS. The expressions of ZO-1, ZO-2, Occludin, Claudin-5 proteins decreased after cerebral ischemia-reperfusion. All drugs inhibited the decrease of ZO-1, except AST I V, and increased Occludin expression; Borneol + AST IV + PNS also up-regulated ZO-2, and the increase in ZO-1, ZO-2, Occludin was greater than that of each drug alone and AST IV + PNS. Conclusions Borneol, AST IV and PNS can relieve the increase of BBB permeability, reduce brain edema and antagonize brain injury to various degrees after cerebral ischemia-reperfusion, which are enhanced by the combination of three drugs. The mechanism may be related to the synergistic inhibition of the down-regulation of ZO-1, ZO-2 and Occludin and the protection of BBB after cerebral ischemia.

Effect of diabetes mellitus on adenosine postconditioning-induced reduction of myocardial ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective To investigate the effect of diabetes mellitus (DM) on adenosine postcondi-tioning-induced reduction of myocardial ischemia-reperfusion (I∕R) injury in rats. Methods Adult male Sprague-Dawley rats, weighing 230-260 g, were used in the study. Type 2 DM was induced by high-fat diet and intraperitoneal l％ streptozocin 35 mg∕kg and confirmed by fasting blood glucose concentration>16. 7 mmol∕L 72 h later. Eighteen rats with type 2 DM were divided into 3 groups (n= 6 each) using a ran-dom number table: sham operation group (DS group), I∕R group (DI∕R group) and adenosine postcondi-tioning group (DAP group). Eighteen healthy nondiabetic rats were selected and randomly divided into 3 groups (n= 6 each): sham operation group (NS group), I∕R group (NI∕R group) and adenosine postcon-ditioning group (NAP group). Myocardial I∕R was induced by 30 min occlusion of the left anterior descend-ing branch of coronary artery followed by 2 h of reperfusion. Venous blood samples were collected from the femoral vein at 2 h of reperfusion for measurement of plasma cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) concentrations (by enzyme-linked immunosorbent assay). The rats were then sacrificed im-mediately after blood sampling for determination of the myocardial ischemic area and infarct size. Results The plasma cTnI and CK-MB concentrations were significantly increased, and the percentage of myocardial infarct size was increased after myocardial I∕R in nondiabetic and diabetic rats. Adenosine postconditioning significantly decreased plasma cTnI and CK-MB concentrations and percentage of myocardial infarct size in nondiabetic and diabetic rats (P<0. 05). Compared with group NAP, the plasma concentrations of cTnI and CK-MB were significantly increased, and the percentage of myocardial infarct size was increased in group DAP (P<0. 05). Conclusion DM can weaken cardioprotection induced by adenosine postcondition-ing in rats.

Efficacy of adductor canal block combined with infiltration anesthesia for postoperative analgesia in patients undergoing total knee arthroplasty / 中华麻醉学杂志

Objective To evaluate the efficacy of adductor canal block combined with infiltration anesthesia for postoperative analgesia in the patients undergoing total knee arthroplasty. Methods Sixty pa?tients of both sexes, aged 65-80 yr, weighing 40-80 kg, of American Society of Anesthesiologists physi?cal statusⅠ or Ⅱ, scheduled for elective unilateral total knee arthroplasty, were divided into 3 groups ( n=20 each) using a random number table: single?injection adductor canal block + infiltration anesthesia group ( group A) , single?injection femoral nerve block+infiltration anesthesia group ( group F) , and infil?tration anesthesia group ( group I) . Ultrasound?guided adductor canal block and femoral nerve block were performed with 0.5% ropivacaine 20 ml before induction of anesthesia in A and F groups, respectively. Af?ter completion of the block, all the patients were ventilated through the laryngeal mask airway under general anesthesia. After installation of the knee prosthesis, local infiltration anesthesia was conducted with 0.2%ropivacaine 50 ml around the knee joint. Acetaminophen oxycodone capsule was taken orally one pill every 6 h starting from the morning on 1st day after surgery. When visual analogue scale ( VAS) score > 5, tram?adol 100 mg was injected intramuscularly as rescue analgesic. At 4, 8, 24, 48 and 72 h after surgery, VAS scores at rest and during activity were recorded, the quadriceps strength was measured, and the re?quirement for analgesic drugs and development of adverse reactions were recorded. Results Compared with group I, VAS scores at rest and during activity were significantly decreased at 4, 8, and 24 h after surger?y, and the consumption of tramadol was significantly decreased after surgery in A and F groups ( P<0.05) . The quadriceps strength at 4 and 8 h after surgery was significantly higher in A and I groups than in group F ( P<0.05) . No patients developed serious adverse reactions in the three groups. Conclusion Adductor ca?nal block combined with infiltration anesthesia provides reliable efficacy for postoperative analgesia with little influence on the quadriceps strength in the patients undergoing total knee arthroplasty.

Autophagy in cerebral ischemia and the effects of traditional Chinese medicine / 中西医结合学报

Autophagy is a lysosome-mediated degradation process for non-essential or damaged cellular constituents, playing an important homeostatic role in cell survival, differentiation and development to maintain homeostasis. Autophagy is involved in tumors as well as neurodegenerative, cardiovascular and cerebrovascular diseases. Recently, active compounds from traditional Chinese medicine (TCM) have been found to modulate the levels of autophagy in tumor cells, nerve cells, myocardial cells and endothelial cells. Ischemic stroke is a major cause of neurological disability and places a heavy burden on family and society. Regaining function can significantly reduce dependence and improve the quality of life of stroke survivors. In healthy cells, autophagy plays a key role in adapting to nutritional deprivation and eliminating aggregated proteins, however inappropriate activation of autophagy may lead to cell death in cerebral ischemia. This paper reviews the process and the molecular basis of autophagy, as well as its roles in cerebral ischemia and the roles of TCM in modulating its activity.