Da Chaihu decoction is a classic prescription for the treatment of cholecystitis that is widely used in clinical practice, and has a definite curative effect. However, due to its diverse components and complex functions, the traditional indexes fail to capture its overall efficacy. Therefore, this study analyzed and predicted the quality markers (Q-markers) of Da Chaihu decoction based on specific chromatogram and network pharmacology to provide a reference for the comprehensive control of the quality. The study obtained 35 potential practical components of Da Chaihu decoction through virtual screening. The specific chromatogram of 15 batches of Da Chaihu decoction was established by HPLC-DAD with neohesperidin as a reference. Compared with the chromatographic peaks and the reference substance, the chemical components were assigned to predict the nine components of albiflorin, paeoniflorin, naringin, hesperidin, neohesperidin, baicalin, wogonoside, saikosaponin b₂, saikosaponin b₁ as Q-markers of Da Chaihu decoction. Finally, the network of the "components-key targets-signal pathways-biological processes" was constructed by network pharmacology to explore the mechanism of Da Chaihu decoction in treating cholecystitis to clarify the accuracy of Q-markers. The results indicated that potential Q-markers could act on multiple targets to regulate inflammatory and metabolism, and then combine to treat cholecystitis. Q-markers could combine with the pharmacologic action of Da Chaihu decoction, which could elucidate the overall efficacy of Da Chaihu decoction. This study explored the Q-markers of Da Chaihu decoction combined with the specific chromatogram and network pharmacology, which provided a basis for the quality control and evaluation of Da Chaihu decoction.

Objective: To explore the effect of moxibustion at Shenque (CV 8) on myocardial structure and function in exercise-induced fatigue rats. Methods: A 12-week treadmill running training was performed to create an exercise-induced fatigue rat model. Sixty eligible male specific-pathogen-free grade Sprague-Dawley rats were randomly divided into a blank group, a control group, a model group, a non-meridian non-acupoint group, a Zusanli (ST 36) group and a Shenque (CV 8) group, with 10 rats in each group. Rats in the blank group did not receive treadmill running training or moxibustion. Rats in the control group did not receive treadmill running training but received mild moxibustion at Shenque (CV 8). Rats in the model group received treadmill running training but no moxibustion. Rats in the non-meridian non-acupoint group, the Zusanli (ST 36) group and the Shenque (CV 8) group received moxibustion at the non-meridian non-acupoint points, Zusanli (ST 36) or Shenque (CV 8) immediately after each treadmill running training, 15 min each time, once a day for 5 consecutive days a week at a 2-day interval, 60 times of moxibustion in total. Left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular diastolic volume (LVDv), left ventricular systolic volume (LVSv), ejection fraction (EF), stroke volume (SV), early diastolic peak flow velocity of mitral valve (E) and late diastolic peak flow velocity of mitral valve (A) of each group before and after the last treadmill running training were measured. Blood was collected 6 h after the last treadmill running training, and serum C-reactive protein (CRP), myoglobin (Mb), creatine kinase-myocardial band (CK-MB), cardiac troponin I (cTnI) and cardiac troponin T (cTnT) levels were detected. Finally, the heart was separated, the heart mass (HM) was measured, the cTnT level of the myocardial tissue was detected, the ultrastructural changes of the left ventricular myocardium were observed by transmission electron microscope, the left ventricular fraction shortening (LVFS), E/A and heart mass index (HMI) were calculated. Results: Compared with the same group before treatment, the rat cardiac LVEDd, LVESd, LVDv, LVSv, SV, E and A were significantly increased (all P<0.01), and the rat LVFS, E/A and EF were significantly decreased (all P<0.01) in the model group and the non-meridian non-acupoint group after treatment; the rat cardiac SV, LVDv, LVSv, E and A were all increased (all P<0.01), while E/A and EF were decreased (all P<0.01) in the Zusanli (ST 36) group after treatment; the rat cardiac LVDv, E and A were significantly increased (P<0.01 or P<0.05), and E/A was significantly decreased (P<0.01) in the Shenque (CV 8) group after treatment. After treatment, compared with the blank group, the rat cardiac LVEDd, LVESd, SV, LVDv, LVSv, E, A, the serum CRP, Mb, CK-MB, cTnI, cTnT and HMI, and the myocardial cTnT were increased (all P<0.01), and the LVFS, E/A and EF were all reduced (all P<0.01) in the model group; compared with the model group and the non-meridian non-acupoint group, rats in the Zusanli (ST 36) group and the Shenque (CV 8) group showed decreased LVEDd, LVESd, SV, LVDv, LVSv, E, A, serum CRP, Mb, CK-MB, cTnI, cTnT and HMI, and myocardial cTnT (P<0.01 or P<0.05), along with increased LVFS, E/A and EF (all P<0.01); compared with the Zusanli (ST 36) group, Mb and A of the Shenque (CV 8) group were decreased (both P<0.01), while both E/A and EF were increased (P<0.01, P<0.05). Transmission electron microscopy examination showed that myofibrils in the blank group and the control group were neatly arranged with clear light and dark bands; the model group and the non-meridian non-acupoint group showed different degrees of myofibril disintegration and breakage, increased and aggregated mitochondria of different sizes, and increased electron density. The myofibrils in the Shenque (CV 8) group and Zusanli (ST 36) group were arranged neatly with clear light and dark bands, and compensatory hyperplasia of mitochondria. Conclusion: Moxibustion at Shenque (CV 8) and Zusanli (ST 36) both can effectively improve the occurrence of myocardial remodeling in exercise-induced fatigue rats, and the effect of moxibustion at Shenque (CV 8) is better in improving cardiac function.

Objective:To explore the effect of Tuina of Three Handing-Three Points on the recovery of motor function, the expression of neuregulin (NRG) 1 and human epidermal growth factor receptor (ErbB) 2 in the injured point of sciatic nerve and L_4-6 spinal cord, and the morphological change of myelin sheath at the injured point of sciatic nerve of rats. Methods:A total of 76 male Sprague-Dawley rats were randomly divided into normal group, sham operation group, model group and Tuina group with 19 rats in each group. The right side sciatic nerve was clamped to make model in the model group and Tuina group. The sham operation group exposed sciatic nerve only. Tuina group received Tuina on Yinmen (BL37), Chengshan (BL57) and Yanglingquan (GB34) with dialing, plucking and kneading using Tuina technique simulator. All of them were tested with Oblique Plate Test before modeling, seven days and 28 days after modeling. Western blotting was used to detect the protein expression of NRG1 and ErbB2 in the injured point of sciatic nerve and L_4-6 spinal cord. The change of myelin sheath at the sciatic nerve injury point was observed and analyzed by transmission electron microscope. Results:Seven days and 28 days after modeling, the scores of Oblique Plate Test were lower in the model group and Tuina group than in the normal group and the sham operation group (P < 0.05); 28 days after modeling, the scores was higher in Tuina group than in the model group (P < 0.05). At the sciatic nerve injury point, three days after modeling, the expression of NRG1 and ErbB2 was higher in the model group and Tuina group than in the normal group and the sham operation group (P < 0.05); seven days and 28 days after modeling, there was no significant difference in NRG1 among groups (P > 0.05); 28 days after modeling, the expression of ErbB2 was higher in the model group and Tuina group than in the normal group and the sham operation group (P < 0.05). In L_4-6 spinal cord, three days after modeling, the expression of NRG1 and ErbB2 was higher in the model group and Tuina group than in the normal group and sham operation group (P < 0.05); seven days after modeling, the expression of NRG1 was higher in the model group and Tuina group than in the sham operation group (P < 0.05), and the expression of ErbB2 was higher in the model group and Tuina group than in the normal group and the sham operation group (P < 0.05); 28 days after modeling, the expression of NRG1 was higher in Tuina group than in the model group (P < 0.05), and there was no significant difference in ErbB2 among groups (P > 0.05). The electron microscope showed that, 28 days after modeling, the myelin sheath collapsed seriously in the model group; while the ultrastructure of the nerve injury point improved, and the myelin sheath of the nerve fiber was relatively intact in Tuina group; the g-ratio value was lower in the model group than in the sham operation group (P < 0.05), the g-ratio value was higher in Tuina group than in the model group (P < 0.05), and no difference was found in g-ratio value between Tuina group and sham operation group (P > 0.05). Conclusion:Three Handing-Three Points could improve the motor function of hind limbs in rats with sciatic nerve injury, which may be related to the adjustment of NRG1 and ErbB2 in the sciatic nerve and spinal cord, to maintain normal myelin sheath structure.

Objective:To investigate the effect of Tuina of Three Handing-Three Points on the motor function of hind limbs, the proliferation of Schwann cell, recovery of myelin sheath and the expression of transforming growth factor (TGF)-β1/Smad2 pathway protein in injured sciatic nerve of rats. Methods:A total of 66 male Sprague-Dawley rats were randomly divided into sham operation group (n = 22), model group (n = 22) and observation group (n = 22). The sciatic nerve injury model was made by clamping method. On the eighth day after modeling, the observation group received stimulation on Yinmen (BL37), Chengshan (BL57) and Yanglingquan (GB34). The sciatic functional index (SFI) was measured before intervention and 21 days after intervention. The Oblique Plate Test was performed before intervention, and seven days, 14 days and 21 days after intervention. The expression of S100, TGF-β1 and Smad2 were observed by immunofluorescence. The expression of TGF-β1, Smad2 and p-Smad2 was detected by Western blotting. Results:Before intervention, SFI was lower in the model group and observation group than in the sham operation group (P < 0.05); 21 days after intervention, SFI and the angle of Oblique Plate Test were higher in the observation group than in the model group (P < 0.05). Immunofluorescence showed that, 21 days after intervention, the expression of S100 was lower in the model group than in the sham operation group (P < 0.01), and was higher in the observation group than in the model group (P < 0.05), no difference was found between the observation group and the sham operation group (P > 0.05). Western blotting showed that, before intervention and seven days after intervention, the expression of TGF-β1, Smad2 and p-Smad2 were higher in the model group than in the sham operation group; 21 days after intervention, no difference was found in the expression among groups (P > 0.05) Conclusion:Three Handing-Three Points could promote the proliferation of Schwann cell and the recovery of myelin sheath, to improve the motor function of hind limbs in rats with sciatic nerve injury, which may not be related to TGF-β1/Smad2 pathway.

Objective:To investigate the mechanism of Three Handing-Three Points on pain function in sciatic nerve injury rats by observing the changes of chemokine (C-X3-C motif) ligand 1, CX3CL1)/chemokine (C-X3-C motif) receptor 1 (CX3CR1) protein and mRNA expression in spinal dorsal horn. Methods:A total of 74 male Sprague-Dawley rats were randomly divided into normal group (n= 12), sham group (n = 24), model group (n = 25), and Three Handing-Three Points group (Tuina group,n = 13). The model group and Tuina group prepared the sciatic nerve injury model. The sham group exposed sciatic nerve only. Tuina group received Tuina on Yinmen (BL37), Chengshan (BL57) and Yanglingquan (GB34) with Tuina manipulation emulator. The photothermal pain threshold was measured seven days after modeling and after 20 days of intervention; cumulative pain score was measured seven days after modeling, and after ten days and 20 days of intervention. The spinal dorsal horn tissues were extracted to detect the protein and mRNA expression of CX3CL1/CX3CR1 with Western blotting and RT-PCR seven days after modeling and after 20 days of intervention. The microglia morphology in spinal dorsal horn was observed with immunofluorescence after 20 days of intervention. Results:Seven days after modeling, compared with the normal group, the photothermal pain tolerance threshold increased in the model group and the sham group (P < 0.05); compared with the sham group, the cumulative pain score increased in the model group and Tuina group (P < 0.05). After ten days of intervention, the cumulative pain score was lower in Tuina group than in the model group (P < 0.05). After 20 days of intervention, both the photothermal pain tolerance threshold and cumulative pain score were lower in Tuina group than in the model group (P < 0.05). There was no significant difference in the expression of CX3CL1/CX3CR1 protein and mRNA on the seven days after modeling and after 20 days of intervention (P > 0.05). The microglia in the model group were partially activated or completely activated, while those in Tuina group were unactivated or partially activated after 20 days of intervention. Conclusion:Three Handing-Three Points can improve the pain function of sciatic nerve injured rats, which may associate with regulating microglia through the pathway other than CX3CL1/CX3CR1.

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

BACKGROUNDY-27632 is a specific inhibitor of Rho-associated coiled kinase (ROCK) and has been shown to promote the survival and induce the differentiation of a variety of cells types. However, the effects of Y-27632 on adult human adipose tissue-derived stem cells (ADSCs) are unclear. This study aimed to investigate the effects of Y-27632 on the neuronal-like differentiation of ADSCs.

METHODSADSCs were isolated from women undergoing plastic surgery and cultured. ADSCs were treated with different doses of Y-27632 and observed morphological changes under microscope. The expression of nestin, neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2) in ADSCs treated with Y-27632 was detected by immunocytochemistry and Western blotting analysis.

RESULTSY-27632 had the potency to induce neuronal-like differentiation in ADSCs in a dose-dependent manner. Moreover, the differentiation induced by Y-27632 was recovered upon drug withdraw. ADSCs treated with Y-27632 expressed neuronal markers such as NSE, MAP-2 and nestin while untreated ADSCs did not express these markers.

CONCLUSIONSelective ROCK inhibitor Y-27632 could potentiate the neuronal-like differentiation of ADSCs, suggesting that Y-27632 could be utilized to induce the differentiation of ADSCs to neurons and facilitate the clinical application of ADSCs in tissue engineering.

Adipose Tissue ; cytology ; Adult ; Amides ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Humans ; Neurons ; cytology ; Pyridines ; pharmacology ; Stem Cells ; cytology ; drug effects

Objective To investigate the clonal types of Staphylococcus aureus collected from 2004to 2010 in patients with blood stream infection from a Grade A tertiary care hospital in Shanghai as well as the dynamic changes and to detect the variation in antimicrobial resistance and virulence-gene content in different strain types.Methods A total of 103 nonduplicate S.aureus isolates were collected from 2004 to 2010 from inpatients with S.aureus blood stream infection from Shanghai Huashan hospital.Determination of oxacillin MICs and the type of SCCmec gene were used to screen MRSA.Typing of S.aureus isolates was identified by using multilocus sequence typing(MLST) and S.aureus-specific staphylococcal protein A typing(spa typing),PCR was used to detect the antimicrobial resistance and virulence-gene.Results Sixtysix isolates(64.1%) MRSA were detected in 103 nonduplicate S.aureus isolates,and 35 isolates were MRSA with SCCmec type Ⅱ ,Twenty-nine isolates were MRSA with SCCmec type Ⅱ,two isolates were MRSA with SCCmec type Ⅳ,Thirty-seven isolates(35.9%) were MSSA.Thirty-three MRSA isolates were ST5,Twenty-nine MRSA isolates were ST239,two MRSA isolates were ST59,one MRSA isolates was ST641 and one MRSA isolates was ST6.All of the other clones belonged to MSSA.The percentage of ST5 and ST239 were decreased significantly after 2009(ST5 was decreased from 52.9% to 15.4%; ST239 was decreased from 61.1% to 15.4%),and new clonal types MSSA increased significantly(in 2009,the percentage of ST7 was 41.7%; new clonal types such as ST188 and ST15 were detected in 2010).In 2010,it was shown that 84.6% of MSSA were isolated from S.aureus blood stream infection,nineteen isolates(18.4%) harbored mupA gene and 41 isolates(39.8%) harbored qacA/B gene in 103 nonduplicate S.aureus isolates.It was shown that 70.6% ST239 harbored qacA/B gene.Four isolates of ST398 and 1 isolates of ST9were detected which were originally from animal.There was no significant difference of the virulence gene presence in the same strain types except sasX、lukSF and arcA genes,but there were a lot of genes which were restricted to different genomic background.Conclusions The percentage of ST5 and ST239 were decreased and new clonal types of MSSA were increased significantly in S.aureus blood stream infection,antimicrobial resistance and virulence-gene were restricted to different clonal types.

ObjectiveTo investigate the distribution and effect on antibiotic resistance of the novel cell wall anchored protein-encoding gene sasX.MethodsA total of 300 S.aureus isolates were randomly collected from inpatients with S.aureusinfection in ShanghaiHuashanhospitalin 2004, 2007and 2010.Meanwhile,170 S.aureus isolates from the nasal swabs of healthy people were collected as part of a population-based community prevalence study.Typing of S.aureus isolates were identified by using multilocus sequence typing (MLST) and S.aureus-specific staphylococcal protein A typing (spa typing).Determination of oxacillin MICs was used to screen MRSA.PCR and sequencing were used to analyze sasX gene.The effect on antibiotic resistance of sasX gene was detect by disc agar diffusion drug sensitive test.ResultsThe major clonal types of the 300 S.aureus isolates collected from inpatients with S.aureus infection were ST239 ( 110,36.7％) and ST5 (122,40.7％).From 2004 to 2010,the percentage of isolates from inpatients with S.aureus infection was increased from 17％ to 39％,but sasX was only found in 0.59％ of the S.aureus isolates from the nasal swabs of healthy people.The percentage of sasX positive was increased from 47.2％ to 83.8％ in ST239.The percentage of sasX positive MRSA was increased from 26.4％ to 50.8％,but the percentage of sasX positive MSSA was about 10％.Antibiotic resistance of sasX positive strains were higher than that of sasX negative strains.Conclusions SasX gene is mainly detected in nosocomial pathogenic S.aureus and it is a possible virulence factor of S.aureus in hosptal setting.The presence of sasX gene is related to antibiotic resistance.For better understanding the real function of this novel gene,further studies such as expression of the encoded protein should be carried out.

Objective To observe the function of recombinant human tissue factor pathway inhibitor1(rTFPI-1)in acute myocardial infarction in rabbit. Method Forty New Zealand White rabbits were subjected to coronary artery occlusion for 120 min and followed by reperfusion for 60 min,then they were ranlow dose rTFPI-1 group(n=10/group).The extent of ischemic area and the extent of myocardial infarction area were measured by Evan's blue stain and TTC stain,respectively.The degrees of infarction severity and ischemic severity were expressed as the ratios of the total left ventrieular wall area.The degrees of infarction severity and ischemic severity in different groups were compared by using one-way ANOVA and then followed by LSD procedure.Results The degree of infarction severity in the larger dose rTFPI-1 group was significantly lessened than that in low dose RTFPI-1 group and control group(P<0.001),and than that in modcrate dose rTFPI-1 group as well(P<0.05).The degree of infarction severity in the moderate dose rTFPI1 group was significantly lessened than that in low dose rTFPI-1 group and control group(P<0.001).There was no significant difference in degree of infarction severity between low dose rTFPI-1 group and control group(P>0.05).Conclusions Human rTFPI-1 might decrease myocardial infarction severity and save the survival myocardial tissue.