Bushen Huoxue Prescription Regulates PINP and β-CTX in Treatment of Femoral Head Necrosis with Syndrome of Liver and Kidney Deficiency / 中国实验方剂学杂志

ObjectiveTo explore the therapeutic mechanism of Bushen Huoxue prescription from the perspective of bone metabolism by observing the clinical efficacy of this prescription in treating femoral head necrosis （ONFH， syndrome of liver and kidney deficiency） and its influences on bone metabolism indexes： N-terminal propeptide （PINP） and β-collagen degradation product （β-CTX）. MethodSixty-six ONFH patients with the syndrome of liver and kidney deficiency in Zhengzhou Traditional Chinese Medicine Hospital of Orthopedics from December 2021 to September 2022 were selected. The patients were randomized into an experimental group and a control group by the parallel control method， with 33 patients in each group. The experimental group received Bushen Huoxue prescription orally， while the control group received Xianlinggubao Capsules orally， with a treatment cycle of 6 months. The visual analogue scale （VAS） score， Harris score， Association Research Circulation Osseous （ARCO） staging， imaging changes， quantitative scores of TCM symptoms， and serum levels of PINP and β-CTX were determined before and after treatment. The occurrence of adverse events and reactions was recorded. ResultThe total response rate in the experimental group was 83.87% （26/31）， which was higher than that （68.75%， 22/32） in the control group （Z=-2.096， P<0.05）. After treatment， the single and total scores of TCM symptoms， VAS score， and β-CTX level decreased in the two groups （P<0.05）. Moreover， the decreases in the scores of hip pain， lower limb mobility， soreness of waist and knees， and lower limb flaccidity， total score of TCM symptoms， VAS score， and β-CTX level in the experimental were larger than those in the control group （P<0.05）. After treatment， the imaging results showed no significant improvement in the two groups. The Harris score and PINP level in both groups increased after treatment （P<0.05）， and the increases were more obvious in the experimental group than in the control group （P<0.05）. No serious adverse event or adverse reaction appeared during the observation period. ConclusionBushen Huoxue prescription can relieve pain and TCM symptoms and improve the hip joint function in treating ONFH patients with the syndrome of liver and kidney deficiency. It can inhibit the development of ONFH， increase PINP， and decrease β-CTX. No obvious side effect appears during the clinical observation period， which shows that Bushen Huoxue prescription has good safety.

Effect of Jianpi Bushen Huoxue Prescription on Rat Brain Microvascular Endothelial Cells Based on HIF-1α/VEGF Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of Jianpi Bushen Huoxue prescription （JPBSHX） on rat brain microvascular endothelial cells （RBMECs） based on hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor （VEGF） signaling pathway， aiming to provide a theoretical basis for the treatment of ischemic stroke. MethodTwelve 8-week-old male SPF-grade SD rats were selected. Eight of them were randomly chosen and given 3.25 g·mL-1 JPBSHX solution by gavage at a dose of 10 mL·kg-1 for 5 consecutive days to prepare the medicated serum， which was then preserved for later use. The remaining four rats were given the same volume of normal saline. Follow-up operations were the same as those of the above eight rats. Normal rat serum was collected and stored for later use. RBMECs were revived， cultured， passaged， and randomly divided into five groups： normal group （20% normal rat serum+80% high glucose DMEM）， model group （hypoxia-reoxygenation injury） （20% normal rat serum+80% glucose-free DMEM）， medicated serum group （20% JPBSHX-medicated serum+80% glucose-free DMEM）， medicated serum+HIF-1α inhibitor group （20% JPBSHX-medicated serum+HIF-1α inhibitor 1 mg +80% glucose-free DMEM）， and medicated serum+VEGF inhibitor group （20% JPBSHX-medicated serum +VEGF inhibitor 1 mg+80% glucose-free DMEM）. The relative protein expression levels of Claudin-1 and Claudin-5 in RBMECs， the expression levels of HIF-1α and VEGF in RBMEC culture supernatants， the repair ability of RBMECs， and the number of nodes， microvessels， and their lengths after 72 h of culture were observed in each group. ResultAfter 24 h of reoxygenation， the scratch healing rate in the model group was significantly lower than in the normal group （P<0.01）. Compared with the result in the model group， the scratch healing rates significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group （P<0.05）. However， the healing rates in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group were significantly lower than that in the medicated serum group （P<0.05）. The number of nodes， microvessels， and total length of microvessels in the model group were significantly lower than those in the normal group （P<0.01）. These indicators significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group compared with those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group compared with those in medicated serum group （P<0.05）. The relative expression levels of Claudin-1 and Claudin-5 proteins were significantly lower in the model group than in the normal group （P<0.01）. These levels were significantly higher in medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. The expression levels of HIF-1α and VEGF in the RBMEC culture supernatants were significantly lower in the model group than those in the normal group （P<0.01）. These levels were significantly higher in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. ConclusionJPBSHX can promote the proliferation， migration， and angiogenesis， such as tubule formation， of RBMECs damaged by hypoxia-reoxygenation injury， and this effect may be achieved through the regulation of the HIF-1α/VEGF signaling pathway.

Bushen Huoxue Prescription Replenishes Diminished Ovarian Reserve in Rats Through PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the mechanism of Bushen Huoxue prescription in regulating the related factors in phosphatidylinositol 3-kinase （PI3K）/ protein kinase B （Akt）/ mammalian target of rapamycin （mTOR） pathway and improving ovarian reserve function of rats with diminished ovarian reserve （DOR）. Method:Sixty DOR model rats were duplicated by Ataya method （intraperitoneal injection of cyclophosphamide） and then randomized into the model group， estradiol valerate （0.000 9 g·kg^-1） group， and high- （33 g·kg^-1）， middle- （16.5 g·kg^-1） and low-dose （8.25 g·kg^-1） Bushen Huoxue prescription groups， with 12 rats in each group. Another 12 healthy rats were classified into the blank control group. The rats in each group were given the corresponding drugs by gavage， while those in the blank control group and model group received the same volume of normal saline， once per day， for 14 successive days. After treatment， the ovarian tissue was stained with hematoxylin-eosin （HE） for observing the changes in quantities of primary follicles， mature follicles， and total follicles under a light microscope， followed by the detection of vascular endothelial growth factor （VEGF） expression in the ovarian tissue by immunohistochemistry. The protein expression levels of PI3K， Akt， mTOR， and cysteine-dependent aspartate-directed protease-3 （Caspase-3） in the ovarian tissue were assayed by Western blot， whereas the mRNA expression levels of PI3K， Akt， and mTOR were measured by Real-time polymerase chain reaction （Real-time PCR）. Result:As revealed by comparison with the blank control group， the quantities of mature follicles and total follicles in the ovarian tissue of model group were significantly reduced （<italic>P</italic><0.05， <italic>P</italic><0.01）. The protein expression levels of VEGF and Caspase-3 in the ovarian tissue were increased （<italic>P</italic><0.05）， while the protein and mRNA expression levels of PI3K， Akt， and mTOR were decreased （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， Bushen Huoxue prescription at the high and middle doses elevated the quantities of mature follicles and total follicles to varying degrees （<italic>P</italic><0.05， <italic>P</italic><0.01）， and VEGF increased most significantly in the middle-dose Bushen Huoxue prescription group （<italic>P</italic><0.05）. Caspase-3 in the low-， middle-， and high-dose Bushen Huoxue prescription groups and the western medicine group declined. The protein and mRNA expression levels of PI3K， Akt， and mTOR were up-regulated in the middle- and high-dose Bushen Huoxue prescription groups （<italic>P</italic><0.05， <italic>P</italic><0.01）， and the levels in the middle-dose Bushen Huoxue prescription group were closer to those in the blank control group. Conclusion:Bushen Huoxue prescription effectively improves the ovarian reserve function of rats with DOR and increases the number of follicles possibly by up-regulating VEGF expression in the ovarian tissue， activating the PI3K/Akt/mTOR signaling pathway， and regulating the content of Caspase-3.

Clinical Research on Bushen Huoxue Prescription in Treating Kidney Deficiency and Blood Stasis Type Primary Osteoporosis / 中国中医药信息杂志

Objective To observe the efficacy and safety of Bushen Huoxue Prescription in the treatment of kidney deficiency and blood stasis type primary osteoporosis. Methods Totally 50 patients with kidney deficiency and blood stasis type primary osteoporosis were collected. They were given Bushen Huoxue Prescription granules twice a day, taking with water, for 12 weeks. Before and after treatment, the femoral neck, Ward's area, L1–4 bone mineral density (BMD) were measured. VAS and TCM syndrome integrals were evaluated before and 4, 8 and 12 week of treatment. The level of serum osteocalcin (OC), β-C-terminal telopeptides of type I collagen (β-CTX) and osteoprotegerin (OPG) were detected before and after treatment. Hematuria routine, liver and kidney function were under safety testing before and after treatment. Results Bushen Huoxue Prescription could significantly increase BMD of left femoral neck and Ward's triangle (P=0.001, P=0.044). L1–4 BMD increased, without statistical significance (P=0.172). The total effective rate was 60.870% (28/46) for L1–4 and left femoral neck and 58.696% (27/46) for Ward's triangle. The VAS and TCM syndrome integral decreased significantly 4 weeks treatment compared with that before the treatment (P<0.01), and the scores decreased significantly with the prolongation of treatment time (F=159.690, P<0.001; F=163.970, P<0.001). After 12 weeks, treatment, the total effective rate for TCM syndromes was 78.26% (36/46). Serum levels of OC and β-CTX showed a tendency to increase, while serum levels of OPG showed a tendency to decrease, without statistical significance (P=0.087, P=0.091, P=0.419). There was no serious adverse reaction in hematuria routine and liver and kidney function. Conclusion Bushen Huoxue Prescription has obvious clinical efficacy for kidney deficiency and blood stasis type primary osteoporosis, which can improve the patients' BMD, improve bone metabolism, relive bone pain, with high level of safety.

Effects of Bushen Huoxue Prescription on Granulosa Cell Apoptosisi of Premature Ovarian Failure Mice / 中国中医药信息杂志

Objective To discuss Bushen Huoxue Prescription mechanism of action on follicular granulosa cell apoptosis of premature ovarian failure (POF) mice. Methods The mouse zona pellucida 3 as the antigen, multi-point injection of subcutaneous immunization BALB/c to female mice was used to establish autoimmune POF model. Forty clean female mice were randomly divided into blank group, model group, Estradiol Valerate group and Bushen Huoxue Prescription group. All medicine groups were given relevant medicine for gavage, while model and blank groups were given normal saline with the same amount for gavage for 15 days. After the treatment, ovary, uterus, and thymus were weighed, and the viscera index was calculated. An ovarian slice was taken and HE staining was used to observe granulosa cell apoptosis. The levels of E2, LH, and FSH were detected by using the radioimmunoassay method. RT-PCR was used to analyze Fas and Fas-L mRNA expressions in ovarian granulosa cells. Results Compared with blank group, the weight of ovary, thymus, and uterus decreased significantly (P<0.05, P<0.01);Serum E2 level dropped (P<0.01);levels of FSH and LH, and expressions of Fas and Fas-L mRNA increased significantly (P<0.01). Compared with model group, granulosa cell apoptosis significantly decreased, and ovary index, uterus index, and thymus index increased (P<0.05);serum E2 level increased (P<0.01);levels of FSH and LH, and expressions of Fas and Fas-L mRNA significantly decreased (P<0.05). Conclusion Bushen Huoxue Prescription can decrease expressions of Fas and Fas-L mRNA to inhibit cell apoptosis by adjusting the hormone level of hypothalamus-pituitary-ovarianaxis.