OBJECTIVE To investigate the effects and potential mechanism of paeoniflorin on oxidative stress of ulcerative colitis （UC） mice based on adenosine monophosphate-activated protein kinase （AMPK）/nuclear factor-erythroid 2-related factor 2 （Nrf2） pathway. METHODS Male BALB/c mice were randomly divided into control group， model group， inhibitor group （AMPK inhibitor Compound C 20 mg/kg）， paeoniflorin low-， medium- and high-dose groups （paeoniflorin 12.5， 25， 50 mg/kg）， high- dose of paeoniflorin+inhibitor group （paeoniflorin 50 mg/kg+Compound C 20 mg/kg）， with 8 mice in each group. Except for the control group， mice in all other groups were given 4% dextran sulfate sodium solution for 5 days to establish the UC model. Subsequently， mice in each drug group were given the corresponding drug solution intragastrically or intraperitoneally， once a day， for 7 consecutive days. The changes in body weight of mice were recorded during the experiment. Twenty-four hours after the last administration， colon length， malondialdehyde （MDA） content， and activities of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） in colon tissues were measured； histopathological morphology of colon tissues， tight junctions between intestinal epithelial cells， and histopathological scoring were all observed and evaluated； the mRNA expressions of AMPK and Nrf2， as well as the protein expressions of heme oxygenase-1（HO-1）， occludin and claudin-1， were all determined in colon tissue. RESULTS Compared with model group， paeoniflorin groups exhibited recovery from pathological changes such as inflammatory cell infiltration and crypt damage in the colon tissue， as well as improved tight junction damage between intestinal epithelial cells. Additionally， significant increases or upregulations were observed in body weight， colon length， activities of SOD and GSH-Px， phosphorylation level of AMPK， and protein expression of Nrf2， HO-1， occludin， claudin-1， and mRNA expressions of AMPK and Nrf2； concurrently， MDA content and histopathological scores were significantly reduced （P＜ 0.05 or P＜0.01）. In contrast， the inhibitor group showed comparable （P＞0.05） or worse （P＜0.05 or P＜0.01） indicators compared to the model group. Conversely， the addition of AMPK inhibitor could significantly reverse the improvement of high- dose paconiflorin （P＜0.01）. CONCLUSIONS Paeoniflorin can repair intestinal epithelial cell damage in mice， improve tight junctions between epithelial cells， upregulate the expression of related proteins， and promote the expression and secretion of antioxidant-promoting molecules， thereby ameliorating UC； its mechanism may be associated with activating AMPK/Nrf2 antioxidant pathway.

Objective To investigate the effect of diosgenin on the proliferation and apoptosis of breast cancer cells and its potential molecular mechanism. Methods The breast cancer cell line MCF-7 was treated with low, medium, and high doses of diosgenin, and cell proliferation was detected through the MMT method. Flow cytometry was used to detect cell apoptosis. Nuclear-cytoplasmic-protein separation method was applied to detect the subcellular localization of death associated protein (DAXX). qRT-PCR and Western blot were used to detect the expressions of DAXX and c-Jun N-terminal kinase pathway (JNK)-related proteins. Results Diosgenin considerably inhibited the proliferation of MCF-7 cells and promoted cell apoptosis in a concentration-dependent manner. Diosgenin can promote the movement of DAXX from nucleus into the cytoplasm. Diosgenin upregulated the expression of cell surface death receptor (Fas), increased the phosphorylation levels of JNK and mitogen activated protein kinase (p38), and activated the JNK/p38 signaling pathway with concentration dependence. Conclusion Diosgenin inhibits the proliferation and promotes the apoptosis of the breast cancer cell line MCF-7, whose mechanism may be related to the regulation of DAXX subcellular localization and the activation of JNK/p38 signaling pathway.

ObjectiveTo explore the protective mechanism of paeoniflorin on mice with ulcerative colitis （UC） through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） autophagy pathway. MethodUC mouse model was established by allowing mice freely drink 4% DSS， and 56 BALB/c male mice were randomly divided into model group， AMPK inhibitor group （20 mg·kg^-1）， paeoniflorin （50 mg·kg^-1） + inhibitor （20 mg·kg^-1） group， and high dose （50 mg·kg^-1）， medium dose （25 mg·kg^-1）， and low dose （12.5 mg·kg^-1） paeoniflorin groups. After seven days of drug intervention， the protective effect of paeoniflorin on mice with UC was determined by comparing the body weight， disease activity index （DAI） changes， and Hematoxylin-eosin （HE） staining results. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the serum of mice in each group， and immunofluorescence was utilized to detect microtubule-associated protein 1 light chain 3 （LC3） content in the colon， AMPK， mTOR proteins， and their phosphorylated proteins including p-AMPK and p-mTOR in the colon tissue were detected by Western blot， and the mRNA expression levels of AMPK， mTOR， Beclin1， LC3， and p62 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group showed a decrease in body mass， an increase in DAI score， and severe pathological damage to the colon. The levels of inflammatory factors including TNF-α and IL-6 increased in serum （P<0.01）， while the protein levels of LC3 and p-AMPK/AMPK were down-regulated in colon tissue， and those of p-mTOR/mTOR were up-regulated （P<0.01）. The mRNA expression levels of AMPK and LC3 were down-regulated， while the mRNA expression levels of mTOR and p62 were up-regulated （P<0.01）. Compared with the model group and the paeoniflorin + inhibitor group， the mice treated with paeoniflorin showed an increase in body mass， a decrease in DAI score， a reduction in pathological damage to colon tissue， and a reduction in the levels of inflammatory factors of TNF-α and IL-6 in serum （P<0.05）. The protein levels of LC3 and p-AMPK/AMPK in colon tissue were up-regulated， while the protein levels of p-mTOR/mTOR were down-regulated （P<0.01）. The mRNA expression levels of AMPK， Beclin1， and LC3 were up-regulated， while the mRNA expression of mTOR and p62 were down-regulated （P<0.01）. The colon tissue of the inhibitor group was severely damaged， and the trend of various indicators was completely opposite to that of the high dose paeoniflorin group. ConclusionPaeoniflorin can enhance autophagy and reduce inflammatory damage in mice with UC by activating the AMPK/mTOR signaling pathway and thus play a protective role.

Objective:To evaluate the effect of gastrogin on AMP-activated protein kinase (AMPK)/transient receptor potential anchor protein 1 (TRPA1) signaling pathway in rats with neuropathic pain.Methods:Thirty-six SPF-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-230 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation+ normal saline group (SHAM group), neuropathic pain+ normal saline group (NP group), and neuropathic pain+ gastrogin group (GAS group). Neuropathic pain was induced by chronic constrictive injury to sciatic nerve under 2% isoflurane anaesthesia. The sciatic nerve was only exposed but not ligated in SHAM group. Gastrogin 100 mg/kg was intraperitoneally injected for 14 consecutive days after developing the model in GAS group, while the equal volume of normal saline was given instead in SHAM and NP groups. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before developing the model (T 0) and 1, 3, 5, 7, 10 and 14 days after developing the model (T 1-6). The rats were anesthetized and sacrificed following the measurement of pain thresholds at T 4 and T 6. The lumbar segment (L 4-6) of the spinal cord was removed for determination of TRPA1 mRNA expression (by quantitative real-time polymerase chain reaction), expression of TRPA1, AMPK and p-AMPK (by Western blot), expression of TRPA1 (by immunofluorescence staining) and expression of tumor necrosis-alpha(TNF-α), interleukin-1beta(IL-1β) and c-fos (by immunohistochemistry). Results:Compared with SHAM group, MWT and TWL were significantly decreased at T 1-6, the expression of TRPA1 mRNA, TRPA1, TNF-α, IL-1β and c-fos was up-regulated, the expression of p-AMPK was down-regulated ( P<0.05), and no significant change was found in AMPK expression in NP group ( P>0.05). Compared with NP group, MWT at T 3-6 and TWL at T 2-6 were significantly increased, the expression of TRPA1 mRNA, TRPA1, TNF-α, IL-1β and c-fos was down-regulated, and p-AMPK expression was up-regulated ( P<0.05), and no significant change was found in AMPK expression in GAS group ( P>0.05). Conclusions:The mechanism by which gastrogin reduces neuropathic pain may be related to modulating the expression of the AMPK/TRPA1 signaling pathway in rats.

Objective:To investigate the effects of sleep disorders (SD) on the radiation injury of hematopoietic stem cells (HSCs) in bone marrow (BM).Methods:Totolly 56 C57BL/6J male mice aged 6-8 weeks were enrolled in this study. They were subjected to whole body irradiation of 60Co γ-rays with doses of 5.0 and 7.5 Gy. A SD model was established using a SD device. According to the random number table method, the mice were divided into seven groups: the control group (Con group), the SD group, the mere radiation group (IR group), the group of post-irradiation SD (IR+ SD group), the group of post-irradiation SD treated with phosphate buffer solution (IR+ SD+ PBS group), the group of post-irradiation SD treated with GSK2795039 (IR+ SD+ GSK group), and the group of post-irradiation SD treated with N-acetylcysteine (IR+ SD+ NAC group), with in eight mice each group. The changes in the peripheral blood of the mice after 5.0 Gy irradiation were detected using the collected tail venous blood, and the survival rates of the mice after 7.5 Gy irradiation were observed. The changes in the density and count of bone marrow cells were observed using hematoxylin and eosin (HE) staining. The number of hematopoietic stem cells in bone marrow (LSK cells), as well as their apoptosis level and changes in cell cycle, were detected using flow cytometry. Furthermore, indicators of LSK, such as reactive oxygen species(ROS) and mitochondrial-derived reactive oxygen species (mtROS), were analyzed. Nicotinamide adenine dinucleotide phosphate (NADP+ /NADPH) and glutathione (GSSG/GSH) were detected using an enzyme microplate reader in order to observe the oxidative stress level of LSK. Furthermore, flow cytometry was employed to sort the LSK cells from the mice, and flow cytometry was used to detect the expression of NADPH oxidase 2(NOX2) and cysteinyl aspartate specific proteinnase-1(Caspase-1), and polymerase chain reaction (PCR) was used to detect the expression of inflammatory factors such as NOX1-4, interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 18 (IL-18), and tumor necrosis factor α (TNF-α). Results:Compared to the IR group, the IR+ SD group exhibited significantly slower recovery of white blood cells (WBC) and platelets (PLT) ( t = 4.39, 6.37, P < 0.05), the bone marrow cell count decreasing from (2.14 ± 0.38) × 10 7 to (3.59 ± 0.29) × 10 7 ( t = 8.55, P < 0.05), significantly decreased proportion of G 0-phase LSK cells, significantly increased proportion of apoptotic cells ( t = 7.53, 8.21, P < 0.05), and significantly increased DCFH-DA, MitoSOX, and NADP+ /NADPH ( t = 22.99, 29.47, 3.77, P<0.05). In the case of IR, SD further promoted the activation of NOX2 and led to increases in the mRNA expression of downstream inflammatory factors such as IL-1β, IL-6, IL-18, and TNF-α ( t = 6.95, 6.01, 8.39, 4.91, 5.56, P < 0.05). Inhibition of NOX2-ROS could prevent the SD-induced aggravation of post-irradiation hematopoietic injury. This significantly reduced the apoptotic rate of LSK cells and the expression of inflammatory factors, ultimately accelerating the hematopoietic recovery of LSK cells ( t = 9.24, 3.92, P < 0.05). Conclusions:SD can aggravate the IR-induced injury of hematopoietic stem cells in bone marrow, primarily by activating the NOX2-ROS-Caspase-1 axis. This will increase the levels of intracellular inflammatory factors and ROS, promote cell apoptosis, and ultimately inhibit the hematopoietic recovery of bone marrow.

ObjectiveTo explore the mechanism of Huanglian Jiedutang in inhibiting the pyroptosis mediated by NOD-like receptor protein 3 （NLRP3） inflammasomes and alleviating the acute liver injury （ALI） induced by lipopolysaccharide （LPS） in the mouse model of sepsis. MethodFifty-four male C57BL/6 mice were randomized into blank， model， low- （3.08 g·kg^-1）， medium- （6.15 g·kg^-1）， and high-dose （12.30 g·kg^-1） Huanglian Jiedutang， and positive control （dexamethasone） groups （n=9）. The mice were administrated with Huanglian Jiedutang at different doses by gavage for 7 days， and then LPS （15 mg·kg^-1） was injected intraperitoneally for the modeling of sepsis. In the positive control group， dexamethasone （0.05 g·kg^-1） was injected intraperitoneally 1.5 h after modeling， and the mouse sepsis score （MSS） was recorded 12 h after modeling. The mice were sacrificed for the collection of blood and liver tissue samples. The levels of alanine transaminase （ALT） and aspartate transaminase （AST） were measured by a biochemical analyzer. The levels of tumor necrosis factor （TNF）-α， interleukin （IL）-6， IL-1β， and IL-18 in the serum were measured by enzyme-linked immunosorbent assay kits. Hematoxylin-eosin staining was used to observe the pathological changes in the liver tissue. The content of NLRP3 was observed by the immunofluorescence assay. The expression of apoptosis-associated speck-like protein containing CARD （ASC） was detected by immunohistochemistry. The protein levels of NLRP3， ASC， Caspase-1， and gasdermin D （GSDMD） in the liver tissue were determined by Western blot. Real-time quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of GSDMD， Caspase-1， IL-1β， and IL-18. ResultCompared with the blank group， the model group showed elevated levels of ALT and AST （P<0.01） and risen levels of inflammatory cytokines in the serum （P<0.01）. In addition， the modeling resulted in edema and necrosis in the liver， and up-regulated the protein levels of GSDMD， NLRP3， ASC， and Caspase-1 （P<0.01） and the mRNA levels of GSDMD， Caspase-1， IL-1β， and IL-18 （P<0.01）. Compared with the model group， the drug intervention groups showed reduced content of inflammatory cytokines （P<0.01）， alleviated pathological damage in the liver tissue， and down-regulated protein levels of GSDMD， NLRP3， ASC， and Caspase-1 （P<0.05，P<0.01） and mRNA levels of GSDMD， Caspase-1， IL-1β， and IL-18 （P<0.05，P<0.01） in the liver tissue. ConclusionHuanglian Jiedutang can inhibit pyroptosis and reduce inflammation by inhibiting the activation of NLRP3 inflammasomes， thus demonstrating a therapeutic effect on acute liver injury in the mouse model of sepsis induced by LPS.

Objective:To explore the practical role of massive open online course (MOOC) mode based on the cultivation of creativity and thinking ability in clinical human anatomy teaching.Methods:Two classes of clinical medicine students of Batch 2019 were selected in the study, and one class was control group ( n=73), which adopted the traditional teaching mode of face-to-face teaching; the other class was research group ( n=79) and the MOOC mode based on the cultivation of creativity and thinking ability for teaching was adopted. After the teaching, the students' creativity, thinking ability, self-learning ability and learning interest were compared, and the mastery of knowledge (theoretical scores and anatomical operation assessment) and satisfaction with teaching were compared between the two groups. SPSS 19.0 was used for t test, chi-square test and rank sum test. Results:The scores of creativity, thinking ability, self-study ability and learning interest of the research group were significantly higher than those of the control group after the teaching ( P<0.05). The scores of theoretical knowledge[(91.41±6.28) points] and anatomical operation[(87.41±7.25) points] in the research group were significantly higher than those in the control group after the teaching[(85.24±7.36) points and (80.26±6.38) points] ( P<0.05). There was significant difference in the distribution of teaching satisfaction between the two groups ( P<0.05), and the total satisfaction rate for teaching of the research group (94.94%) was higher than that of the control group (83.56%). Conclusion:The MOOC mode based on the cultivation of creativity and thinking ability in clinical human anatomy teaching can significantly improve students' creativity, thinking ability and self-learning ability, improve their learning interest and mastery of human anatomy knowledge, and improve their satisfaction with teaching.

Nanomaterial-based drug sustainable release systems have been tentatively applied to bone regeneration. They, however, still face disadvantages of high toxicity, low biocompatibility, and low drug-load capacity. In view of the low toxicity and high biocompatibility of polymer nanomaterials and the excellent load capacity of hollow nanomaterials with high specific surface area, we evaluated the hollow polydopamine nanoparticles (HPDA NPs), in order to find an optimal system to effectively deliver the osteogenic drugs to improve treatment of bone defect. Data demonstrated that the HPDA NPs synthesized herein could efficiently load four types of osteogenic drugs and the drugs can effectively release from the HPDA NPs for a relatively longer time in vitro and in vivo with low toxicity and high biocompatibility. Results of qRT-PCR, ALP, and alizarin red S staining showed that drugs released from the HPDA NPs could promote osteogenic differentiation and proliferation of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Image data from micro-CT and H&E staining showed that all four osteogenic drugs released from the HPDA NPs effectively promoted bone regeneration in the defect of tooth extraction fossa in vivo, especially tacrolimus. These results suggest that the HPDA NPs, the biodegradable hollow polymer nanoparticles with high drug load rate and sustainable release ability, have good prospect to treat the bone defect in future clinical practice.
Animals ; Bone Regeneration ; Indoles ; Nanoparticles ; Osteogenesis ; Pharmaceutical Preparations ; Polymers ; Rats

COVID-19 has swept globally and Pakistan is no exception.To investigate the initial introductions and transmissions of the SARS-CoV-2 in Pakistan,we performed the largest genomic epidemiology study of COVID-19 in Pakistan and generated 150 complete SARS-CoV-2 genome sequences from samples collected from March 16 to June 1,2020.We identified a total of 347 mutated positions,31 of which were over-represented in Pakistan.Meanwhile,we found over 1000 intra-host single-nucleotide variants(iSNVs).Several of them occurred concurrently,indicating possible interactions among them or coevolution.Some of the high-frequency iSNVs in Pakistan were not observed in the global population,suggesting strong purifying selections.The genomic epidemiology revealed five distinctive spreading clusters.The largest cluster consisted of 74 viruses which were derived from different geographic locations of Pakistan and formed a deep hierarchical structure,indicating an extensive and persistent nation-wide transmission of the virus that was probably attributed to a signature mutation(G8371T in ORF 1ab)of this cluster.Further-more,28 putative international introductions were identified,several of which are consistent with the epidemiological investigations.In all,this study has inferred the possible pathways of introduc-tions and transmissions of SARS-CoV-2 in Pakistan,which could aid ongoing and future viral surveillance and COVID-19 control.

Objective:To compare the clinical effects of Zero-profile (Zero-p) intervertebral fusion and titanium plate combined with cage fusion in treating symptomatic adjacent segment disease (ASD) after anterior cervical discectomy and fusion (ACDF).Methods:Retrospective analysis was performed on 26 patients who underwent ACDF and readmission due to concurrent symptomatic ASD from October 2014 to June 2019. There were 17 males and 9 females, aged 54.15±8.60 (range 41-68) years. The index level included C 2, 3 1 case, C 3, 4 3 cases, C 4,5 9 cases, C 5, 6 6 cases, C 6, 7 7 cases. Twelve cases underwent anterior cervical decompression with Zero-p intervertebral fusion and fixation (Zero-p group), while 14 cases underwent anterior cervical decompression with titanium plate combined with cage fusion and fixation (titanium plate group). The following parameters, including operative duration, intraoperative blood loss, Japanese Orthopaedic Association (JOA) score, visual analogue score (VAS), neck disability index (NDI), dysphagia Bazaz grade, bone graft fusion Eck grade, C 2-C 7 Cobb angle, and related complications, were compared between the two groups. Results:The operation was performed successfully in all the patients. The patients were followed up for averagely 33.38±21.26 (range 12-71) months. The operation duration was 95.83±5.47 (range 89-105) min in the Zero-p group, which was shorter than 121.28±8.24 (range 106-131) min in the titanium plate group. The Bazaz classification of dysphagia in the Zero-p group was superior to the titanium plate group at 1 month after operation ( W=126.00, P=0.022). Neither group had dysphagia 3 months after surgery. The JOA score increased from preoperative 9.50±1.31 to 14.33±0.78, and VAS decreased from 5.33±1.67 to 0.83±0.72 in the Zero-p group. The NDI decreased from 43.62%±9.31% to 14.99%±3.26%, and C 2-C 7 Cobb changed from 8.26°±2.92° to 14.80°±4.18° in the Zero-p group. The JOA score increased from preoperative 9.14±1.79 to 14.71±0.73, and VAS decreased from 5.43±1.55 to 1.43±0.76 in the titanium plate group. The NDI decreased from 43.76%±8.47% to 14.22%±4.59%, and C 2-C 7 Cobb changed from 5.53°±9.04° to 14.68°±6.89° in the titanium plate group. No complication, such as screw loosening or breakage or esophageal injury, occurred during the follow-up. Conclusion:Both methods can achieve good clinical effects in treating symptomatic ASD and can restore the physiological curvature of the cervical spine to a certain extent. Furthermore, the advantages of Zero-p intervertebral fusion include shorter operation duration, reducing soft tissue injury and less postoperative dysphagia.