Effect of Bushen Yiqi Huoxue Recipe on Placental Vasculature in Pregnant Rats with Fetal Growth Restriction Induced by Passive Smoking

Interactions of vascular endothelial growth factor （VEGF） with receptors VEGFR1/Fltl and VEGFR2/Flk1, and those of angiopoietins （Ang-1, Ang-2） with receptor Tie2 play important roles in placental angiogenesis. This study investigated vascular morphology and expression of these angiogenic factors in rat placenta on the day 15, 18, 21 of gestation （D 15, D 18 and D21）. The rats were randomly assigned into 3 groups： normal group, model group [fetal growth restriction （FGR） model], and Bushen Tqi Huoxue （BYHR） recipe treatment group （BYHR group, the pregnant rats with FGR were treated with BYHR recipe）. Morphological analysis indicated that during initial villous formation, fetal nucle- ated erythrocytes （FNEs） appeared in maternal blood sinus （MBS）. Subsequently, FNEs were sur- rounded by endothelial cells to form fetal capillary （FC） and then by trophoblast cells to form villi. As pregnancy proceeded, FC density increased progressively with increasing endothelial identification staining （EIS） in normal and BYHR groups. Whereas, villous formation was suppressed, normal in- crease in FC density was impaired and EIS was weakened in model group. Quantitative PCR analysis showed that VEGF and Flkl mRNA increased over gestation in all groups, indicating that VEGF might play a pivotal role in FC growth during late gestation. VEGF mRNA was increased on D15, while de- creased on D21 in model group as compared with normal group and BYHR group. Immunohistochemi- cally, Ang-2 protein was highly expressed in FNEs, gradually disappeared as villi matured, and decreased over gestation in all groups, indicating that Ang-2 might play a pivotal role in villous formation, which was further supported by decreased Ang-2 mRNA and protein expression in model group on D 15. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio increased from D15 to D18 in all groups as placenta matured. Ang-1 mRNA, Tie2 mRNA and Ang-1/Ang-2 ratio were decreased on D18 in model group as compared with normal and BYHR groups, indicating delayed maturity of FGR placenta. Alterations in angiogenic factors may result in altered placental vasculature and cause placental insufficiency. BYHR recipe could balance the angiogenic factors to promote the formation and maturation of FGR placental vasculature.

Effect of Huoxue Yiqi recipe on proliferation,migration and tube formation of miR-484 overexpressed umbilical vein endothelial cells

Objective:To explore the possible mechanism of Huoxue Yiqi recipe promoting angiogenesis by targeting VEGF with miR-484.Methods:The model of miR-484 overexpressed in HUVEC was prepared and intervened with Huoxue Yiqi Recipe Freeze-dried powder.The effects of Huoxue Yiqi Recipe on proliferation,migration and tubular forming ability of HUVEC were separatly detected by CCK-8 method,cell scratch test and Matrigel three-dimensional forming test in vitro.The effect of Huoxue Yiqi Recipe on VEGFA mRNA expression was detected by real-time fluorescence quantitative PCR.Results:Compared with the control group,the proliferation and migration area of cells in miR-484 overexpression model group decreased,the length,number of branches and nodes involved in tube formation decreased,the tube formation ability decreased significantly,and the expression level of VEGFA mRNA decreased,with statistical significance.Compared with the model group,the cell proliferation ability,migration area,length,number of branches and nodes involved in tube formation,tube formation ability and expression level of VEGFA mRNA in the Huoxue Yiqi Prescription group were up-regulated,and the difference was statistically significant(P<0.05).Conclusion:MiR-484 inhibits the expression of VEGFA and the proliferation,migration and formation of HUVEC,and Huoxue Yiqi recipe has a certain negative regulation effect on this process,which may be a new target of Huoxue Yiqi recipe to promote angiogenesis.

Therapeutic Effect of Bushen Huoxue Recipe(补肾活血方) on Autoimmune Premature Ovarian Failure Mice Established by Immunization with Recombinant Porcine Zona Pellucida 4 Antigen

Objective： To investigate the efficacy and mechanism of Bushen Huoxue Recipe （补肾活血方, BHR） in the treatment of mudne autoimmune premature ovarian failure （POF）. Methods： The recombinant porcine zona pellucida 4 （pZP4） was expressed in E. coli BL21 （DE3） strain within prokaryotic plasmid pET28a （＋）, purified by Ni-affinity chromatography and verified by Western blot. Murine autoimmune POF model was established by immunization with pZP4 of female BALB/c mice. Fifty POF mice were randomly divided into 5 groups, which were respectively given low （3.75 mg/kg）, moderate （7.5 mg/kg）, and high dose （15.0 mg/kg） of BHR by gastrogavage once daily for 20 days, with 17-13-estradiol （0.13 mg/kg） and normal saline as positive and negative control. Estrous cycles were analyzed through vaginal smears, serum estradiol （E2） levels, and anti-pZP4 antibody titers were detected by ELISA. The proliferative responses in vitro of spleen lymphocytes to pZP4 antigen restimulation were measured by [3H]-thymidine incorporation, and the histomorphology changes of ovary were evaluated by optical microscope. ]Results： The purified pZP4 was visible as a single lane with 14.4 kD in SDS-PAGE and Western blot. The murine POF model with lengthening estrous cycles, decreased levels of serum E2, high titers of serum anti-pZP4 antibody, and reduced ovarian follicles and corpus lutea were established by immunization with recombinant pZP4. Treatment with moderate and high dosage BHR significantly increased ovarian follicles and reduced the proliferation of spleen lymphocytes to the pZP4 antigen of POF mice （P〈0.05）. However, only the high dosage BHR administration significantly improved the estrous cycles, elevated the serum E2 levels （P〈0.01）, and decreased the serum anti-pZP4 antibody titers of model mice （P〈0.05）. Conclusions： The recombinant pZP4 could evoke the antigen-specific immune response in mice and induce the autoimmune ovarian injury. It has been demonstrated that BHR was able to increase the serum E2 levels and protect ovarian functions from the autoimmune injury in murine POF model.

Effects of Yiqi Huoxue Recipe （益气活血方） and Coxsackie Virus B Type 3 on the Expression of Ribosomal Protein S20 in Rat Cardiac Myocytes

Objective： To study the effects of Yiqi Huoxue Recipe （益气活血方） and Coxsackie B virus type 3 （CVB3） on the expression of ribosomal protein S20 in rat cardiac myocytes, to explore the pathogenesis of myocarditis induced by CVB3 and the mechanism of Yiqi Huoxue Recipe on gene level, and to further investigate whether Yiqi Huoxue Recipe is an effective prescription for CVB3 myocarditis. Methods： A modified suppression subtractive hybridization （SSH） was used to isolate differentially expressed genes between the CVB3 infection group and the treatment group with Yiqi Huoxue Recipe. The results were further verified by fluorescence RT-PCR. Results： The results of SSH showed that the gene expression of ribosomal protein S20 in the treatment group was higher than that in the CVB3 infection group （P〈0.05）, which agreed with the results of fluorescent RT-PCR. Conclusion： Down-regulation of ribosomal protein S20 mRNA expression might be one of the mechanisms in CVB3 myocarditis, and Yiqi Huoxue Recipe could achieve the treatment of viral myocarditis by regulating the expression of ribosomal protein S20.

Clinical Observation on Effect of Yiqi Huoxue Recipe (益气活血方) in Comprehensive Treatment on Advanced Stage of Esophageal Cancer

Objective: To observe the effect of Yiqi Huoxue (replenishing Qi and activating blood circulation, YQHX) recipe in patients with advanced stage of esophageal cancer treated with radiotherapy and intervention chemotherapy. Methods:In comparing 31 patients who were treated with radiochemotherapy (RCT, Group A), and 31 patients treated with RCT plus YQHX recipe(Group B). Results:Immediate effective rates were 48.4% in Group A and 64.5% in Group B (P>0.05). The function of bone marrow in Group B was obviously better than that in Group A. The 1 year metastasis rate in Group A was higher than that in Group B, but the longterm survival rate of Group B was obviously higher than that in Group A (P<0.05). Conclusion: YQHX recipe could reduce the bone marrow suppression caused by RCT, lower the metastasis rate, prolong the longterm survival and improve the quality of life.

Multicenter Randomized Double-Blind Controlled Clinical Study of Huoxue Tongluo Recipe(活血通络方)External Washing in the Treatment of Chemotherapy-Induced Peripheral Neuropathy

Objective: To objectively evaluate the efficacy of Huoxue Tongluo Recipe(活血通络方) in the treatment of chemotherapy-induced peripheral neuropathy(CIPN).Methods: A multicenter, randomized, double-blind, block, and parallel-controlled study was conducted in which 184 subjects who met the inclusion criteria were randomized into a trial group(92 cases), a control group(46 cases), a placebo group(46 cases) at a 2:1:1 ratio, respectively.Huoxue Tongluo Recipe(活血通络方) granules, Huangqi Guizhi Wuwu Decoction(黄芪桂枝五物汤) instant granules, placebo instant granules were given for hand and foot soaking, with water temperature 38 ℃-45 ℃, soaking for 20 minutes and the intervention time was 14 days.Results:(1) The total effective test drug was 41.30%, the control group was 32.61%, and the placebo group was 32.61%.There was no significant difference in the efficiency of the 3 groups by CMH chi-square test(P>0.05);(2) In the statistics of previous chemotherapy regimens including oxaliplatin or paclitaxel as stratification factors, the overall efficiency of the trial group was significantly different from the other 2 groups.Conclusion: Huoxue Tongluo Recipe(活血通络方) external washing can improve the peripheral nerve injury caused by chemotherapy to some extent, especially in patients with previous chemotherapy regimen including oxaliplatin and paclitaxel.

Huoxue Jiedu Huayu recipe(活血解毒化瘀方)alleviates contralateral renal fibrosis in unilateral ureteral obstruction rats by inhibiting the transformation of macrophages to myofibroblast

OBJECTIVE:To investigate the action and underlying mechanisms of Huoxue Jiedu Huayu recipe(活血解毒化瘀方,HJHR)against unilateral ureteral obstruction(UUO)-induced injury in the contralateral kidney.METHODS:Forty-eight male Sprague-Dawley rats weighing(200±10)g were used in this study and randomly assigned to 4 groups:a sham group,a UUO group,a UUO+eplerenone(EPL)group,and a UUO+HJHR group.The contralateral kidneys were harvested for further study 180 d after surgery.Histological analysis,immunohistochemistry and immunofluorescence were used to study the fibrosis of the contralateral kidneys obtained from UUO rats.Contralateral kidney damagerelated pathway proteins were detected by real-time polymerase chain reaction and Western blot analysis.RESULTS:HJHR significantly inhibited fibrosis of the contralateral kidney in UUO rats by attenuating the UUOinduced macrophage-to-myofibroblast transition(MMT)in the contralateral kidney.Moreover,HJHR attenuated fibrosis in the contralateral kidney of UUO rats by preventing MMT through the aldosterone/mineralocorticoid receptor/serum/glucocorticoid regulated kinase 1 pathway.CONCLUSIONS:Our findings suggest that HJHR may be a potential treatment for renal interstitial fibrosis of obstructive nephropathy.

Huoxue Anxin Recipe(活血安心方)Promotes Myocardium Angiogenesis of Acute Myocardial Infarction Rats by Up-Regulating miR-210 and Vascular Endothelial Growth Factor

Objective： To investigate the microR NAs（miR NAs） expression profile of acute myocardial infarction（AMI） rats and the regulating effects of Huoxue Anxin Recipe（活血安心方, HAR） on angiogenesis-related miR NAs and genes. Methods： Forty-five Wistar rats were randomly assigned to 3 groups according to a random number table： sham, AMI, and AMI＋HAR groups（15 in each group）. AMI rats were established by ligation of the left descending coronary artery. HAR was intragastrically administered to rats of the AMI＋HAR group for successive 21 days since modeling, meanwhile the same volume of 0.9% normal saline was administered to rats of the sham and AMI groups. Doppler echocardiography was used for noninvasive cardiac function test. Hematoxylin and eosin staining was used to observe the histopathological change. miR NAs expression profile was detected by quantitative realtime polymerase chain reaction（qR T-PCR）. The mR NA and protein expressions of vascular endothelial growth factor（VEGF）, and a target gene of miR-210 was further detected by qR T-PCR and Western blot, respectively. The microvessels density of myocardium was evaluated by CD31 immunostaining. Results： Compared with the sham group, ejection fraction（EF） and fractional shortening（FS） values were decreased significantly in the AMI group（P〈0.01）, while the infarction area and the interstitial collagen deposition were increased obviously. As for the AMI＋HAR group, EF and FS values were increased significantly（P〈0.05 vs. AMI group）, and the infarction area was reduced and the interstitial collagen deposition were alleviated significantly. Total of 23 miR NAs in the AMI group expressed differently by at least 1.5 folds compared with those in the sham group; 5 miR NAs in the AMI＋HAR group expressed differently by at least 1.5 folds compared with those in the AMI group. Among them, miR-210 was low in the AMI group and high in the AMI＋HAR group. The relative mR NA and protein expressions of VEGF were decreased significantly in the AMI group（P〈0.05 vs. sham group）, and increased significantly in the AMI＋HAR group（P〈0.01 vs. AMI group）. CD31 expression area and optical intensity were decreased significantly in the AMI group（P〈0.05 vs. sham group）, and increased significantly in the AMI＋HAR group（P〈0.01 vs. AMI group）. Conclusions： HAR could reduce the infarction area, alleviate the interstitial fibrosis and improve the cardiac function of AMI rats. Those effects could be related to promoting myocardium angiogenesis of HAR by up-regulating miR-210 and VEGF.

Clinical and Experimental Study on Prevention and Treatment of Acute Radiation Injury in Nasopharyngeal Carcinoma Patients by Yiqi Huoxue Yangyin Recipe (益气活血养阴方)

Objective: To observe the effect of Chinese herbal medicine in alleviating acute toxic adverse effect of radiotherapy and on growth of stromal cells in bone marrow. Methods: Seventy two patients with nasopharyngeal carcinoma (NPC) were randomly divided into two groups. Radical radiotherapy was applied to both groups. In the radiotherapeutic period, to the 36 patients in the treated group, Yiqi Huoxue Yangyin Recipe (YHYR) was given additionally by oral taking and compressing on radiation area, and to the 36 patients in the control group, vitamin B 12 solution was given for gargling or compressing. Experimental study was carried out in rats of two groups, irradiated with 5.0 Gy X ray and treated with YHYR or normal saline intraperitoneally, to observe the colony forming unit fibroblastoid (CFU F) in cultured bone marrow stromal cells (BMSC), taken from the rats at different time.Results: The oral mucomembranous radiation damage occurred in 47.2% of the treated group when the dose of radiation reached to 41.4±9.4 Gy, while in the control group, it was 91.7% when the dose reached to 30.9±8.9 Gy. The skin radiation damage occurrence rate in the two groups was 13.9% and 33.3% respectively when the dose of radiation reached to 50.2±5.6 Gy and 43.2±6.3 Gy respectively. Comparison of the two groups showed significant difference ( P <0.01), the radiation damage was significantly slighter in the treated group. Experimental study showed that the rats in the two groups were significantly different in occurrence and degree of bone marrow function inhibition, P <0.01. The numbers of CFU F in cultured BMSC of radiation damaged rats taken at respective different culture time were also significantly different between the two groups, P < 0.01 .Conclusion: YHYR could significantly reduce acute radiation damage of mucomembrane and skin, alleviate the inhibition on bone marrow function, and eliminate the injury of radiation on BMSC.