This study explored whether Astragalus polysaccharide(APS)can regulate the VEGF/VEGFR signaling pathway to affect the proliferative activity of rat intestinal mucosal microvascu-lar endothelial cells(RIMMVECs).RIMMVECs were isolated from newborn rats,then purified and treated with APS at concentrations of 0.1,1.0,10.0,100.0,1 000.0,and 10 000.0 mg/L.MTT was used to determine the effect of APS on RIMMVECs proliferation and screen for the optimal concentration of APS.Subsequently,flow cytometry was used to detect the changes in cell cycle to evaluate the stage of action of APS on the cell cycle in RIMMVECs.Then,the ELISA was used to detect the changes of VEGFA in cell supernatant to evaluate the potential of cell proliferation and angiogenesis.The changes in fluorescence intensity of Fluo-8AM was observed using fluorescence microscopy to evaluate intracellular Ca2+levels.Finally,Western blot was used to detect the ex-pression of PERK in RIMMVECs to analyze the possible mechanism of APS.The results showed that 100 mg/L APS significantly enhanced the proliferative activity of RIMMVECs,increased the content of VEGFA in the cell supernatant,the intracellular Ca2+levels,and the expression of PERK protein,indicating that APS promotes the proliferation of RIMMVECs,which may be a-chieved by promoting the expression of VEGFA and activating the ERK pathway.

This study explored whether Astragalus polysaccharide(APS)can regulate the VEGF/VEGFR signaling pathway to affect the proliferative activity of rat intestinal mucosal microvascu-lar endothelial cells(RIMMVECs).RIMMVECs were isolated from newborn rats,then purified and treated with APS at concentrations of 0.1,1.0,10.0,100.0,1 000.0,and 10 000.0 mg/L.MTT was used to determine the effect of APS on RIMMVECs proliferation and screen for the optimal concentration of APS.Subsequently,flow cytometry was used to detect the changes in cell cycle to evaluate the stage of action of APS on the cell cycle in RIMMVECs.Then,the ELISA was used to detect the changes of VEGFA in cell supernatant to evaluate the potential of cell proliferation and angiogenesis.The changes in fluorescence intensity of Fluo-8AM was observed using fluorescence microscopy to evaluate intracellular Ca2+levels.Finally,Western blot was used to detect the ex-pression of PERK in RIMMVECs to analyze the possible mechanism of APS.The results showed that 100 mg/L APS significantly enhanced the proliferative activity of RIMMVECs,increased the content of VEGFA in the cell supernatant,the intracellular Ca2+levels,and the expression of PERK protein,indicating that APS promotes the proliferation of RIMMVECs,which may be a-chieved by promoting the expression of VEGFA and activating the ERK pathway.

OBJECTIVE: To investigate the effects of tanshinone IIA on apoptosis and autophagy induced by hypoxia/reoxygenation in H9C2 cardiomyocytes and its mechanism. METHODS: H9C2 cardiomyocytes in logarithmic growth phase were divided into control group, hypoxia/reoxygenation model group and tanshinone IIA low-dose, medium-dose and high-dose groups (50, 100, 200 mg/L tanshinone IIA were treated after hypoxia/reoxygenation respectively). The dose with good therapeutic effect was selected for follow-up study. The cells were divided into control group, hypoxia/reoxygenation model group, tanshinone IIA+pcDNA3.1-NC group and tanshinone IIA+pcDNA3.1-ABCE1 group. The cells were transfected with the overexpressed plasmids pcDNA3.1-ABCE1 and pcDNA3.1-NC and then treated accordingly. Cell counting kit-8 (CCK-8) was used to detect H9C2 cell activity in each group. The apoptosis rate of cardiomyocytes was detected by flow cytometry. The ATP-binding cassette transporter E1 (ABCE1), apoptosis-related proteins Bcl-2 and Bax, caspase-3, autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3II/I) and p62 mRNA expression level of H9C2 cells in each group were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expression levels of the above indexes in H9C2 cells were detected by Western blotting. RESULTS: (1) Cell activity and ABCE1 expression: tanshinone IIA inhibited the activity of H9C2 cells induced by hypoxia/reoxygenation, and the effect was significant at medium-dose [(0.95±0.05)% vs. (0.37±0.10)%, P < 0.01], mRNA and protein expression of ABCE1 were significantly reduced [ABCE1 mRNA (2^-ΔΔCt): 2.02±0.13 vs. 3.74±0.17, ABCE1 protein (ABCE1/GAPDH): 0.46±0.04 vs. 0.68±0.07, both P < 0.05]. (2) Expression of apoptosis-related proteins: medium-dose of tanshinone IIA inhibited the apoptosis of H9C2 cells induced by hypoxia/reoxygenation [apoptosis rate: (28.26±2.52)% vs. (45.27±3.07)%, P < 0.05]. Compared with the hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated the protein expression of Bax and caspase-3 in H9C2 cells induced by hypoxia/reoxygenation, and significantly up-regulated the protein expression of Bcl-2 [Bax (Bax/GAPDH): 0.28±0.03 vs. 0.47±0.03, caspase-3 (caspase-3/GAPDH): 0.31±0.02 vs. 0.44±0.03, Bcl-2 (Bcl-2/GAPDH): 0.53±0.02 vs. 0.37±0.05, all P < 0.05]. (3) Expression of autophagy-related proteins: compared with the control group, the positive rate of LC3 in the hypoxia/reoxygenation model group was significantly increased, while the positive rate of LC3 in the medium-dose of tanshinone IIA group was significantly decreased [(20.67±3.09)% vs. (42.67±3.86)%, P < 0.01]. Compared with hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated Beclin-1, LC3II/I and p62 protein expressions [Beclin-1 (Beclin-1/GAPDH): 0.27±0.05 vs. 0.47±0.03, LC3II/I ratio: 0.24±0.05 vs. 0.47±0.04, p62 (p62/GAPDH): 0.21±0.03 vs. 0.48±0.02, all P < 0.05]. (4) Expression of apoptosis and autophagy related proteins after transfection with overexpressed ABCE1 plasmid: compared with tanshinone IIA+pcDNA3.1-NC group, the protein expression levels of Bax, caspase-3, Beclin-1, LC3II/I and p62 in tanshinone IIA+pcDNA3.1-ABCE1 group were significantly up-regulated, while the protein expression level of Bcl-2 was significantly down-regulated. CONCLUSIONS 100 mg/L tanshinone IIA could inhibit autophagy and apoptosis of cardiomyocytes by regulating the expression level of ABCE1. So, it protects H9C2 cardiomyocytes injury induced by hypoxia/reoxygenation.
Humans ; Apoptosis ; ATP-Binding Cassette Transporters/metabolism* ; Autophagy ; bcl-2-Associated X Protein/metabolism* ; Beclin-1/metabolism* ; Caspase 3/metabolism* ; Follow-Up Studies ; Myocytes, Cardiac ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/metabolism* ; Cell Hypoxia

Objective:To observe the surgical method and clinical efficacy of applying calf tissue flap combined with artificial bone of antibiotics loaded calcium sulphate in treatment of tibia osteomyelitis.Methods:From July 2018 to January 2021, calf tissue flaps combined with artificial bone of antibiotics loaded calcium sulphate (or mixed with iliac bone) were applied to treat 16 cases with tibia osteomyelitis in the Department of Hand and Microsurgery of Baoji Third Hospital. There were 10 males and 6 females, aged 15 to 64 years old, with a mean age of 41 years old. For the 5 cases with acute osteomyelitis caused by wound infection, local dressing changes and drainage or VSD wound management were applied after debridement, together with primary systemic anti-infection treatment. After the acute infection period had been under control and stabilised, the wounds were then thoroughly exposed and cavities were filled and covered with the surgical reconstruction procedure with antibiotics-loaded artificial bone of calcium sulphate in combination with calf tissue flaps. For the 11 cases with chronic and hypotoxicity osteomyelitis, calf tissue flaps combined with antibiotics-blended artificial bone of calcium sulphate were applied to fill the cavity and cover the wound in phase I surgical reconstruction after thorough debridement. For the 7 cases with large bone defects or larger cavities after debridement, a mixed bone grafts of antibiotics-loaded artificial bone of calcium sulphate and autologous iliac bone were employed, with muscle flaps or myocutaneous flaps for an embedding repair. Sizes of the tissue flaps were 2.0 cm×3.5 cm to 12.0 cm×23.0 cm. Clinical outcomes were evaluated through follow-ups at outpatient clinic. The therapeutic effect was evaluated by the method described by McKee et al.Results:Except for 1 case of distal necrosis of tissue flap and survived after dressing change, the other tissue flap survived successfully. Postoperative follow-ups lasted for 12 to 40(mean 18) months. All the osteomyelitis were successfully cured, except 1 that had recurrence of osteomyelitis 1 year later, and treated with antibiotics-loaded artificial bone of calcium sulphate combined with autologous iliac bone implants after thorough debridement, and then healed well. The shape and texture of flaps were good. Protective sensations were restored to vary levels after 6 months. The calf regained weight-bearing and walking functions at 1 year after surgery. According to McKee et al., the therapeutic effect was evaluated: 11 cases were cured, 4 cases were improved, and 1 case relapsed, with an effective rate of 93.8%.Conclusion:Application of calf tissue flap combined with antibiotics-loaded artificial bone of calcium sulphate in the treatment of tibia osteomyelitis has a high cure rate and remarkable efficacy. It can significantly reduce the number of surgeries and shorten the course of disease.

Objective:To evaluate the efficacy of a calf tissue flap combined with antibiotic-loaded calcium sulfate (artificial bone or mixed iliac bone graft) in the treatment of foot and ankle osteomyelitis.Methods:A retrospective study was conducted to analyze the 11 cases of foot and ankle osteomyelitis which had been treated at Department of Hand and Microsurgery, The Third Hospital of Baoji from October 2018 to October 2021. There were 8 males and 3 females, aged (42.3±23.7) years. The chronic hypotoxic osteomyelitis was repaired and reconstructed after thorough debridement at one stage with a calf tissue flap combined with antibiotic-loaded calcium sulfate artificial bone to fill the cavity and cover the wound. The acute infected trauma was repaired and reconstructed after thorough debridement at the second stage with a calf tissue flap combined with antibiotic-loaded calcium sulfate artificial bone to fill the cavity and cover the wound only after the acute infection was controlled by local dressing, drainage or negative pressure therapy and systemic anti-infection treatment at the primary stage. The flap size ranged from 3.5 cm × 2.0 cm to 12.0 cm × 6.0 cm. Four cases were treated by a peroneal artery perforator flap combined with antibiotic-loaded calcium sulfate artificial bone, 3 cases by a flap with peroneal artery perforator and peroneal nerve trophic vessel combined with antibiotic-loaded calcium sulfate artificial bone, 3 cases by a posterior tibial artery perforator flap combined with antibiotic-loaded calcium sulfate artificial bone, and one by a peroneus longus muscle flap combined with antibiotic-loaded calcium sulfate artificial bone. Postoperatively, the flap survival, bone union time, ankle function and complications were observed; the therapeutic efficacy was evaluated by comparing infection control indexes at the final follow-up [clinical manifestations like local redness, swelling, pain, ulceration, and exudation, and white blood cell count (WBC), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and calcitoninogen (PCT)].Results:All the flaps survived except for one which developed necrosis at the distal 1/3 of the flap but responded to dressing change. All the patients were followed up for (22.6±11.5) months. The 6-month follow-up revealed that all the flaps were fine in shape and texture. Re-examinations showed that WBC, CRP, ESR and PCT were normal or close to normal, the local skin was free of redness, swelling or ulceration, and protective sensation was restored to varying degrees. X-ray at (12.1±2.3) months showed that lesions disappeared, bony union was achieved, the ankle joint regained basic flexion and extension, and the affected limb also regained weight-bearing and walking functions in all the patients but one whose X-ray at 18 months showed poor bony union but no other symptoms or signs.Conclusion:In the treatment of foot and ankle osteomyelitis, a calf tissue flap combined with antibiotic-loaded calcium sulfate artificial bone can promote bone healing and restore the function of the foot and ankle because it not only fills the cavity and covers the wound but also effectively controls the infection.

Two female patients (patient 1, 22-year-old; patient 2, 50-year-old) received IV infusion of ribavirin injection (4 g in the first dose and the next day 1.2 g thrice daily), oral 2 lopinavir and ritonavir tablets twice daily, and aerosol inhalation of recombinant human interferon α2b for injection for novel coronavirus pneumonia. There was no obvious abnormality in blood routine and liver function before treatment. Laboratory tests showed red blood cell count (RBC) 2.89×10 12/L, hemoglobin (Hb) 75 g/L, alanine aminotransferase (ALT) 22.8 U/L, aspartate aminotransferase (AST) 33.9 U/L, total bilirubin (TBil) 71.2 μmol/L, and indirect bilirubin (IBil) 63.5 μmol/L in patient 1 on the 2nd day of treatment, and RBC 3.46×10 12/L, Hb 95 g/L, ALT 17.7 U/L, AST 21.3 U/L, TBil 86.1 μmol/L, and IBil 67.1 μmol/L in patient 2 on the 3rd day of treatment. The direct antiglobulin test was positive, indirect antiglobulin test was negative, and antinuclear antibody test was negative in both patients. They were diagnosed as having acute hemolytic anemia. Con-sidering the relationship to ribavirin, ribavirin was given in reduced dose and then finally discontinued in patient 1, and was discontinued directly in patient 2. On the basis of continued use of the other 2 drugs, both of them were treated with ursodeoxycholic acid. The Hb and bilirubin level of the 2 patients gradually returned to normal.

Two female patients (patient 1, 22-year-old; patient 2, 50-year-old) received IV infusion of ribavirin injection (4 g in the first dose and the next day 1.2 g thrice daily), oral 2 lopinavir and ritonavir tablets twice daily, and aerosol inhalation of recombinant human interferon α2b for injection for novel coronavirus pneumonia. There was no obvious abnormality in blood routine and liver function before treatment. Laboratory tests showed red blood cell count (RBC) 2.89×10 12/L, hemoglobin (Hb) 75 g/L, alanine aminotransferase (ALT) 22.8 U/L, aspartate aminotransferase (AST) 33.9 U/L, total bilirubin (TBil) 71.2 μmol/L, and indirect bilirubin (IBil) 63.5 μmol/L in patient 1 on the 2nd day of treatment, and RBC 3.46×10 12/L, Hb 95 g/L, ALT 17.7 U/L, AST 21.3 U/L, TBil 86.1 μmol/L, and IBil 67.1 μmol/L in patient 2 on the 3rd day of treatment. The direct antiglobulin test was positive, indirect antiglobulin test was negative, and antinuclear antibody test was negative in both patients. They were diagnosed as having acute hemolytic anemia. Con-sidering the relationship to ribavirin, ribavirin was given in reduced dose and then finally discontinued in patient 1, and was discontinued directly in patient 2. On the basis of continued use of the other 2 drugs, both of them were treated with ursodeoxycholic acid. The Hb and bilirubin level of the 2 patients gradually returned to normal.

Objective:To investigate the neuroprotective effect and possible mechanism on rats with low dose Lipopoly -saccharide ( LPS) preconditioning after spinal cord injury.Methods:120 female SD rats were randomly divided into the empty virus (EV) group,LPS+empty virus (LPS+EV) group,Nrf2 interference virus (NIV) group,LPS+Nrf2 interference virus (LPS+NIV) group.The model of traumatic spinal cord injury ( TSCI) was established by the modified Allen′s method,motor function of the rat hind limb was assessed by the Basso Beattie and Bresnahan (BBB) score at 1,3,7,14 and 28 d after the operation.The injured spinal cord tissue samples were harvested at each time ,and the pathological changes of rat spinal cord were observed by HE staining ,the Nissl body and neuron survival index were observed by Nissl staining ,the expressions of Nrf2 and GCLC protein level were detected by immunohis-tochemical staining and Western blot.Results:The rat BBB score of LPS+EV group increased significantly than EV group at 7,14,28 d after operation ( P<0.05 ,P<0.01 );The NIV group between LPS+NIV group have no statistical significance at each time.As compared with EV group:the Nrf2 protein of LPS+EV group was expression increased significantly and Nissl staining showed that the neurons survival index was increased at 1,3 and 7 d(P<0.05,P<0.01);The GCLC protein of LPS+EV group was expression increased significantly at 1-14 d( P<0.05 );HE staining showed that the injured spinal cord pathological changes of LPS +EV group was obviously improved.Conclusion:Low dose lipopolysaccharide preconditioning can accelerate the nerve function recovery on rats with traumatic spinal cord injury ,the mechanism may be regulated by activating the Nrf 2 antioxidant stress pathway.