Effect of Zuoguiwan on Pancreatic Islet Function in Offspring of Maternal Rats with GDM / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Zuoguiwan on pancreatic islet function in offspring of gestational diabetes mellitus （GDM） maternal rat model and explore the mechanisms of Zuoguiwan in improving pancreatic islet function based on postpartum pancreatic regeneration. MethodHealthy female SD rats with normal blood glucose levels were paired with male rats in a 2∶1 ratio and housed together. Pregnancy was confirmed based on vaginal plugs or vaginal smears. The pregnant rats were divided into the following groups： normal group， model group， insulin group （insulin Detemir， 20 U·kg-1）， low-dose Zuoguiwan group （1.89 g·kg-1）， and high-dose Zuoguiwan group （3.78 g·kg-1）. The GDM rat model was induced using streptozotocin in rats except for those in the normal group. The model was confirmed by blood glucose testing in the maternal rats. Except for the normal and model groups， the other groups received daily administration of corresponding treatments. At 21 days after birth， fasting blood glucose （FBG） and fasting serum insulin （FINS） levels were measured in 6 offspring from each group. The homeostasis model assessment of insulin resistance （HOMA-IR） was calculated， and an oral glucose tolerance test （OGTT） was performed on additional 12 offspring from each group. Blood samples were taken from the abdominal aorta of the offspring at postnatal day 22， and enzyme-linked immunosorbent assay （ELISA） was used to measure insulin， glucagon （GC）， pancreatic polypeptide （PPY）， and somatostatin （SS） levels in the serum. Hematoxylin-eosin （HE） staining was performed to observe pathological changes in the pancreatic tissue of the offspring. Immunofluorescence （IF） was used to observe the area and structure of the pancreatic islets. Western blot was used to detect the expression of key proteins involved in the development and functional expression of pancreatic β-cells， namely pancreatic and duodenal homeobox factor 1 （Pdx1）， Nkx6.1， and Glucose transporter 2 （Glut2）. ResultCompared with the normal group， the model group showed significant increases in FBG and FINS levels， and HOMA-IR （P<0.01）. Compared with the model group， the insulin group showed significant decreases in FBG levels and HOMA-IR （P<0.01）， the low-dose Zuoguiwan group showed a significant decrease in FBG levels （P<0.05）， and the high-dose Zuoguiwan group showed significant decreases in FBG and FINS levels， and HOMA-IR （P<0.01）. Compared with the normal group， the model group showed significant increases in OGTT 60-min blood glucose levels and AUC index （P<0.05， P<0.01）. Compared with the model group， the high-dose Zuoguiwan group showed significant decreases in OGTT60-min blood glucose levels and area under the curve（AUC） index （P<0.05， P<0.01）. HE staining of pancreatic tissue showed that compared with the normal group， the model group had a reduced number of islets and a loose arrangement of acinar cells. Compared with the model group， the groups with drug treatment showed increased number of islets and a compact arrangement of acinar cells. Compared with the normal group， the model group had significantly increased levels of insulin， GC， PPY， and SS in the serum （P<0.01）. Compared with the model group， the low-dose and high-dose Zuoguiwan groups and the insulin group showed significantly decreased serum levels of insulin， GC， PPY， and SS （P<0.05， P<0.01）. IF results showed that compared with the normal group， the model group had a significantly lower positive rate of insulin （P<0.05）. Compared with the model group， the low-dose and high-dose Zuoguiwan groups showed a significant increase in the positive rate of insulin （P<0.05）. There was no significant difference in the positive rate of GC among the groups. In terms of the proportion of insulin and GC in individual islets， compared with the normal group， the model group showed a significant decrease in the proportion of insulin （P<0.01） and a significant increase in the proportion of GC （P<0.01）. Compared with the model group， the low-dose and high-dose Zuoguiwan groups showed significantly increased proportion of insulin （P<0.01） and significantly decreased proportion of GC （P<0.01）. Compared with the normal group， the model group showed significantly decreased expression levels of Pdx1， Nkx6.1， and Glut2 proteins in the pancreatic tissue of GDM offspring （P<0.05）. Compared with the model group， the insulin group and the low-dose Zuoguiwan group showed significant increases in the expression levels of Pdx1 and Nkx6.1 proteins in the pancreatic tissue of GDM offspring （P<0.05）， and the low-dose and high-dose Zuoguiwan groups showed significant increases in the expression levels of Glut2 protein （P<0.05）. ConclusionZuoguiwan can promote pancreatic islet development in offspring of GDM maternal rat model， improve pancreatic islet morphology and function， and alleviate insulin resistance. Its mechanism of action may be related to the regulation of Pdx1， Nkx6.1， and Glut2 protein expression in the pancreatic tissue of offspring.

Protection of Zuoguiwan Against Apoptosis of Follicles in Rats Injured by 60Co-γ Rays： Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Zuoguiwan against 60Co-γ ray-induced premature aging of rats based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty sexually mature female SD rats were irradiated with 60Co-γ rays （6.0 Gy， LD40） for 24 h at one time. Then they were randomized into model group， Bujiale group （0.18 g·kg-1·d-1）， Bujiale （0.09 g·kg-1·d-1） + high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， medium-dose Zuoguiwan group （9.45 g·kg-1·d-1）， and low-dose Zuoguiwan group （4.725 g·kg-1·d-1）. The administration （once a day） lasted 21 days. Serum indexes ［follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and estradiol （E2）］ of rats were detected by enzyme-linked immunosorbent assay （ELISA）， and morphological changes of ovarian tissues were observed based on hematoxylin and eosin （HE） staining. The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） and the protein expression of phosphorylated （p）-PI3K， p-Akt， p-mTOR， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in ovarian tissues by Western blot. ResultCompared with normal group， model group demonstrated increase in serum FSH （P<0.01）， decrease in E2 （P<0.05）， and reduction of follicles and luteum in early ovary （P<0.01）. Moreover， the elevation of apoptosis rate of granulosa cells （P<0.01）， down-regulation of p-PI3K， p-Akt， p-mTOR， and Bcl-2 in ovarian tissue， and increase in expression of Bax were also observed in the model group as compared with the normal group （P<0.01）. In comparison with the model group， the administration groups showed rise of the number of early ovarian follicles， decrease in the apoptosis rate of granulosa cells， increase in the expression of p-PI3K， p-Akt， p-mTOR， and Bcl-2， and down-regulation of Bax， particularly the Bujiale + high-dose Zuoguiwan group（P<0.05，P<0.01）. ConclusionZuoguiwan protects radiation-damaged ovary by activating the expression of PI3K/Akt/mTOR protein in ovarian tissue， increasing Bcl-2， and inhibiting the expression of Bax.

Protection of Zuoguiwan Against Apoptosis of Follicles in Rats Injured by 60Co-γ Rays： Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Zuoguiwan against 60Co-γ ray-induced premature aging of rats based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodSixty sexually mature female SD rats were irradiated with 60Co-γ rays （6.0 Gy， LD40） for 24 h at one time. Then they were randomized into model group， Bujiale group （0.18 g·kg-1·d-1）， Bujiale （0.09 g·kg-1·d-1） + high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， high-dose Zuoguiwan group （23.625 g·kg-1·d-1）， medium-dose Zuoguiwan group （9.45 g·kg-1·d-1）， and low-dose Zuoguiwan group （4.725 g·kg-1·d-1）. The administration （once a day） lasted 21 days. Serum indexes ［follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and estradiol （E2）］ of rats were detected by enzyme-linked immunosorbent assay （ELISA）， and morphological changes of ovarian tissues were observed based on hematoxylin and eosin （HE） staining. The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） and the protein expression of phosphorylated （p）-PI3K， p-Akt， p-mTOR， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in ovarian tissues by Western blot. ResultCompared with normal group， model group demonstrated increase in serum FSH （P<0.01）， decrease in E2 （P<0.05）， and reduction of follicles and luteum in early ovary （P<0.01）. Moreover， the elevation of apoptosis rate of granulosa cells （P<0.01）， down-regulation of p-PI3K， p-Akt， p-mTOR， and Bcl-2 in ovarian tissue， and increase in expression of Bax were also observed in the model group as compared with the normal group （P<0.01）. In comparison with the model group， the administration groups showed rise of the number of early ovarian follicles， decrease in the apoptosis rate of granulosa cells， increase in the expression of p-PI3K， p-Akt， p-mTOR， and Bcl-2， and down-regulation of Bax， particularly the Bujiale + high-dose Zuoguiwan group（P<0.05，P<0.01）. ConclusionZuoguiwan protects radiation-damaged ovary by activating the expression of PI3K/Akt/mTOR protein in ovarian tissue， increasing Bcl-2， and inhibiting the expression of Bax.

Effect of Zuoguiwan on Wnt/β-catenin Signaling Pathway in Ovariectomized Osteoporosis Model Rats / 中国实验方剂学杂志

Objective:To observe the effect of Zuoguiwan on bone metabolism and Wnt/<italic>β</italic>-catenin signaling pathway in ovariectomized osteoporotic rats model， and to explore the molecular biological mechanism of Zuoguiwan in the prevention and treatment of osteoporosis. Method:The rat model of postmenopausal osteoporosis was established by bilateral ovariectomy， 60 female SD rats were randomly divided into sham operation group， model group， positive group （estradiol valerate tablet 0.05 mg·kg^-1·d^-1） and low， middle and high dose groups of Zuoguiwan （5.5，11，22 g·kg^-1·d^-1）.After successful establishment of the model in the 13^th week， intragastric administration （<italic>ig</italic>） was given once a day for a total of 12 weeks. After administration， the histomorphological changes of femur in rats were observed by hematoxylin-eosin （HE） staining， the bone mineral density （BMD） and bone mineral content（BMC） of femur were measured by dual energy X-ray apparatus， and the biomechanical properties of bone were measured by MTS Acumen3 biomechanical testing system. The contents of bone alkaline phosphatase （BALP）， bone glaprotein（BGP），estradiol （E₂） ，and tartrate-resistant acid phosphatase （TRAP）， type Ⅰ procollagen N-terminal propeptide （PINP） in serum were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the protein level of Wnt2，<italic>β</italic>-catenin，low density lipoprotein related receptor protein 5 （LRP5） and the phosphorylation level of glycogen synthase kinase-3<italic>β</italic>（GSK-3<italic>β</italic>） in rat tibia. Result:Compared with sham operation group， the maximum load and stiffness of BMD，BMC， in the model group decreased significantly（<italic>P</italic><0.01）， the contents of E₂ and PINP in serum decreased significantly（<italic>P</italic><0.01）， the content of BALP，BGP，TRAP increased significantly（<italic>P</italic><0.01）， the expression levels of Wnt2，p-GSK-3<italic>β </italic>Ser9，LRP5 and <italic>β</italic>-catenin protein in bone tissue decreased significantly（<italic>P</italic><0.01）， the trabecula of femur became thinner and thinner， the number of bone trabeculae decreased. Compared with model group， the maximum load and stiffness of BMD，BMC， in estradiol group and Zuoguiwan group were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the contents of serum E₂ and PINP were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the content of BALP，BGP，TRAP was significantly decreased （<italic>P</italic><0.01）， and the expression level of Wnt2，p-GSK-3<italic>β</italic> Ser9，LRP5， <italic>β</italic>-catenin protein in bone tissue was significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01） ， the trabeculae of femur became thicker， the number increased， the structure was basically clear. Conclusion:Zuoguiwan has a certain preventive and therapeutic effect on osteoporosis in ovariectomized rats， and its mechanism may be related to increasing the level of estrogen， activating Wnt/<italic>β</italic>-catenin signaling pathway， up-regulating the expression of Wnt2 and LRP5 protein， inhibiting the activity of GSK-3<italic>β</italic>， reducing the degradation of <italic>β</italic>-catenin， coordinating the dynamic coupling balance between bone formation and bone resorption， correcting the disorder of bone metabolism and improving bone morphology.

Discussion on Effect of Zuoguiwan on Skin Development of Kidney-deficient Rats Based on Autophagy / 中国实验方剂学杂志

Objective:To study the effect and mechanism of Zuoguiwan on the autophagy of cells in the skin during the development of congenital kidney-deficient mice， and to explore the application value of kidney tonifying and essence filling method in abnormal growth and development. Method:36 rats with a 2∶1 ratio of male to female were paired， and the pregnant rats were stimulated by combined stress method to build a model of congenital kidney-deficient rats. According to different treatment methods， the pregnant rats were divided into the control group， the kidney-deficient group， and the Zuoguiwan low and high dose groups （2， 8 g·kg^-1）. The control group was given normal saline without modeling. Kidney deficiency group was modeled before intragastric administration of normal saline. Zuoguiwan small dose and high dose groups received intragastric administration of Zuoguiwan suspension. 21 days after the birth of mice， the back skin was taken to observe the skin microstructure and morphology of the mice by HE staining and detect the thickness of dermis and epidermis. The gene and protein expression levels of Wingless3a and <italic>β</italic>-catenin in the skin of neonatal rats were measured by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Double immunofluorescence assay was used to detect the skin autophagy indicators Microtubule-associated protein light chain 3 （LC3）and Sequestosome 1 （SQSTM1-p62）. Result:In the control group， the skin layers were clear and the structure was normal， in the kidney deficiency group， the collagen arrangement in the dermis was loose， the skin composition in Zuoguiwan low and high dose groups was basically normal. Compared with the control group， the epidermal layer of the kidney deficiency group was thickened and the dermis was thinned （<italic>P</italic><0.05）. Compared with the kidney deficiency group， the thickness changes of epidermal layer and dermis layer were significantly restored in Zuoguiwan low and high dose groups （<italic>P</italic><0.05）. Compared with the control group， the expression of Wnt3a and <italic>β</italic>-catenin protein and gene in the kidney deficiency group significantly decreased （<italic>P</italic><0.05）. Compared with the kidney deficiency group， Wnt3a and <italic>β</italic>-catenin protein and gene expression increased in Zuoguiwan low and high dose groups （<italic>P</italic><0.05）. At the same time， immunofluorescence assay showed that compared with the control group， the expression of LC3 was up-regulated and the expression of SQSTM1-p62 was down-regulated in the kidney deficiency group， while Zuoguiwan could reverse such abnormal expression （<italic>P</italic><0.05）. Conclusion:Zuoguiwan can down-regulate the autophagy level of skin cells and improve the abnormal skin development of congenital renal deficiency by increasing the expression of Wnt3a and <italic>β</italic>-catenin protein and gene in the skin of neonatal rats.

Effect of Zuoguiwan on Differentiation of CD4-CD8-T Cells in Thymus of Kidney-deficient Offspring Rats / 中国实验方剂学杂志

Objective:To study the effect of Zuoguiwan on the differentiation of CD4-CD8-T cells in thymus during the development of kidney-deficient fetal rats and the application value of kidney and essence-tonifying method, in order to explore the theoretical connotation of "kidney being the origin of congenital constitution". Method:Male and female rats were paired at a ratio of 2∶1, and three stress methods were combined to construct the kidney-deficient model. Pregnant rats were divided into control group, model group, thymosin group (0.003 g·kg-1), and low-dose Zuoguiwan group and high-dose Zuoguiwan group (2,8 g·kg-1). Normal saline was given to the control group， model group was used for modeling and given normal saline by gavage, thymosin group was given thymosin capsule solution by gavage, and low-dose Zuoguiwan group and high-dose Zuoguiwan group were given Zuoguiwan suspension by gavage. Embryos that were 16 days, 19 days and born on the first day (E16, E19, P1) were selected to calculate fetal rat thymus index. Electron microscope was used to observe thymic epithelial cell ultrastructure of fetal rats. Enzyme-linked immunosorbent assay (ELISA) method was used to detect thymus thymosinβ4 (TMSβ4) and thymosinα1 (Tα1) content, fluid cytology detection of fetal rats on the surface of CD4, CD8, CD25, CD44 expression for identifying T cell type. Result:The morphology of thymus epithelial cells in the model group was irregular, the nucleus was deformed, and the chromatin of the nucleus was significantly increased. The results of ELISA showed that the content of TMSβ4 and Tα1 in thymus increased in the model group and the low-dose and high-dose Zuoguiwan groups. Flow cytometry showed that, compared with the control group, CD4-CD8-T cells were significantly increased in the model group at E16, E19 and P1 stages (P<0.05), suggesting that T cells were blocked during the development from DN to DP. CD4-CD8-CD25-CD44-T (DN4) was significantly increased (P<0.05), suggesting that T cell development was inhibited during DN4-DP transition. Compared with the model group, CD4-CD8-T cells were significantly reduced in low and high-dose groups (P<0.05) at E16, E19 and P1 stages. At the same time, DN4 was significantly reduced (P<0.05), suggesting that Zuoguiwan could promote the development of T cells from DN4 stage to DP stage as thymosin. Conclusion:Zuoguiwan can improve the structure of thymus epithelial cells in rats with kidney deficiency, and induce the differentiation of T cells in thymus from DN4 stage to DP stage by increasing the secretion of TMSβ4 and Tα1 in thymus of embryonic rats, indicating that Zuoguiwan can promote the development and maturation of T cells and regulate the immune status of fetal rats with kidney deficiency by tonifying kidney and spermatogenesis.

Clinical Efficacy of Postmenopausal Knee Osteoarthritis with Liver and Kidney Deficiencies Based on Theory of Syndrome Differentiation and Treatment of Kidney / 中国实验方剂学杂志

Objective:To discuss the clinical efficacy of modified Zuoguiwan on postmenopausal osteoarthritis of knee (KOA) with deficiency of liver and kidney based on theory of syndrome differentiation and treatment of kidney, and its effect on endocrine hormone and cartilage metabolism.Method:One hundred and forty patients of KOA were randomly divided into control group (70 cases) and observation group (70 cases) by random number table. Patients in two group got glucosamine hydrochloride capsule for 12 weeks, 1 capsule/time, 2 times/day, and those with obvious pain was added celecoxib capsules for 4 weeks, 0.2 g/time, 1 time/day. The control group took Kang Zengsheng pills orally. patients in observation group was also added with modified Zuoguiwan for 12 weeks, 1 dose/day. Before and after treatment, pain level during activity and rest by visual simulation of pain (VAS) were scored. And visual scale of osteoarthritis index (WOMAC) of Western Ontario and McMaster University, knee osteoarthritis severity index (ISOA), deficiency of liver and kidney, self-rating anxiety scale (SAS) and self-rating depression scale (SDS) were scored. And levels of serum estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), transforming growth factor-β (TGF-β), interleukin-1β (IL-1β), matrix metalloproteinase-3 (MMP-3) and tumor necrosis factor-α (TNF-α) were scored.Result:Scores of VAS during activity and rest in observation group were lower than those in control group (P<0.01). The total score of WOMAC, scores of pain, stiffness, joint function, deficiency of liver and kidney, SAS and SDS were all lower than those in control group (P<0.01). Total score of ISOA, symptoms and signs, maximum walking distance, daily life were lower than those in control group (P<0.01). Levels of E2, FSH, LH, TNF-α, IL-1β and MMP-3 were lower than those in control group (P<0.01). Level of TGF-β was higher than that in control group (P<0.01). And total clinical effective rate in observation group was 89.23%(58/65), which was higher than 74.60%(47/63) in control group (χ2=4.793, P<0.05).Conclusion:Modified Zuoguiwan can obviously alleviate symptoms, improve joint function, regulate the level of endocrine hormone, relieve anxiety and depression, regulate the environment of cartilage metabolism, inhibit inflammatory reaction, and improve the patients' ability of daily life and clinical efficacy.

Effect of Decomposed Zuoguiwan Recipes in Improving BMD of Ovariectomized Rats by Regulating OPG/RANKL Pathway Mediated by β2AR / 中国实验方剂学杂志

Objective:To investigate the mechanism of decomposed Zuoguiwan（ZGW） recipes in treating ovariectomized osteoporosis rats. Method:Forty Sprague-Dawley female rats were equally and randomly divided into Sham-operated group, ovariectomized model group, positive group, and low and high-dose ZGW groups. After 12 weeks of administration by gavage, the bone mineral density (BMD) of rats' distal femur was measured by micro-CT, the morphology of bone tissue were observed by hematoxylin-eosin staining (HE), β-cross-linked c-telopeptide of type Ι collagen (β-CTX) and bone-specific alkaline phosphatase (BALP) in serum were detected by enzyme-linked immunosorbent assay (ELISA), and the mRNA and protein expressions of β2AR, OPG and RANKL were evaluated by Western blot analysis and real-time quantitative polymerase chain reaction （PCR）. Result:Compared with Sham-operated group, BMD of rats in ovariectomized model group was decreased (P<0.01), morphology of bone tissue was destroyed, serum BALP was reduced, while β-CTX was boosted (P<0.01)，mRNA and protein expressions of OPG in tibia were reduced, while RANKL were increased, and mRNA and protein expressions of β2AR in the hypothalamus were decreased (P<0.05, P<0.01). Compared with ovariectomized model group, BMDs of rats in low and high-dose ZGW groups were increased (P<0.01), morphology of bone tissue was repaired, serum BALP and mRNA and protein expressions of OPG in tibia were up-regulated (P<0.05, P<0.01), whereas serum β-CTX and mRNA and protein expressions of β2AR in the hypothalamus and RANKL in tibia were down-regulated (P<0.05, P<0.01). Conclusion:Yang-nourishing components in decomposed Zuoguiwan recipes can improve BMD of ovariectomized rats by regulating OPG/RANKL pathway mediated by β2AR. "Seeking Yin in Yang" is a crucial mechanism of Zuoguiwan in treating ovariectomized osteoporosis in rats.

Effect of Zuoguiwan on Serum Related Indexes and Pancreatic PDX-1 Expression in GDM RatsWANG Chao-qun1，2, WANG Yong-hui2, LIU Tao2, XU Kai-xia2, ZHAO Le2, ZHOU Ran2*（1.HuBei University of Traditional Chinese Medicine（TCM），Wuhan 430065，China；2.Shanxi University of TCM，Taiyuan 030024，China） / 中国实验方剂学杂志

Objective:To investigate the effect of maternal intervention of Zuoguiwan on the improvement of serum and pancreatic index in rats of gestational diabetes mellitus(GDM). Method:The GDM pregnant rats were replicated by monogamous cage and introneal injection of streptozotocin (STZ). The pregnant rats were randomly divided into normal group, model group, insulin group (20 U·kg-1), and low, medium and high-dose Zuoguiwan groups (2.5，5.0，10.0 g·kg-1). The insulin group was given hypodermic injection of portal winter insulin, all drug groups were respectively administrated with corrsponding drugs for 2 weeks, once a day. Meanwhile, control group and model group were given physiological saline by gavage. After that, the levels of serum insulin, fasting plasma glucose（FBG）, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA), protein and gene expression levels of Pancreas-duodenal homologous box factor-1 (PDX-1) were respectively detected by Western blot and real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. Result:Compared with normal group, FBG, TNF-α and IL-6 levels of pregnant rats in model group increased significantly (P<0.01), the level of IL-8 increased uniformly (P<0.05), and serum insulin level, protein and gene expression levels of PDX-1 decreased significantly (P<0.01). Compared with model group, FBG and serum IL-8 levels of pregnant rats in insulin group were significantly lower (P<0.01), while serum insulin level, protein and gene expression levels of PDX-1 increased significantly(P<0.01). FBG, IL-8, IL-6 and TNF-α levels in serum of pregnant rats in medium-dose Zuoguiwan group decreased obviously, meanwhile serum insulin level, protein and gene expression levels of PDX-1 increased significantly (P<0.01). Conclusion:Zuoguiwan may promote the secretion of insulin by reducing the serum inflammatory factors in GDM rats, and at the same time up-regulate the expression of PDX-1 protein and gene, so as to restrain insulin resistance, reduce the damage of pancreatic cells and improve the blood glucose of rats.

Effect of Zuoguiwan on Expression of Ca2+ Transport Pathway-related Protein in Ovariectomized Rats / 中国实验方剂学杂志

Objective::To explore the effect of Zuoguiwan on the bone mineral density (BMD) and the expressions of Ca2+ transport-associated protein in ovariectomized rats. Method::The 48 female SD rats were randomly divided into six groups: normal group, model group, sham operation group, estrogen group(0.167 mg·kg-1) and low and high-dose Zuoguiwan groups(9.6, 38.4 g·kg-1), with 10 rats in each group. Except for the sham-operated group, the ovariectomized rats in the other groups received the bilateral ovariectomy. Therapeutic intervention was given in each group for 3 months after the establishment of the model. After 12 weeks, BMD was measured using dualenergy X-ray absorptiometry. Tartrated presistant acid phosphatse(TRACP) and serum calcium were detected by biochemical kits.Protein expression in Ca2+ transport (Bone tissue) was detected by Western blot. Result::Compared with the normal group, the serum calcium of the model group was decreased(P<0.01). Compared with the normal group, BMD of the model group was decreased (P<0.01). The serum calcium of rats in high-dose group and western medicine group was higher than that of model group(P<0.01). BMD in model group was lower than that of Zuoguiwan groups and estrogen group(P<0.05). There was no significant difference in TRACP among the groups. Nilestriol and Zuoguiwan can down-regulate the expressions of TRPV5, NCX1, CaBP-D28K and PMCA1b in bone tissue of castrated rats(P<0.05, P<0.01). Conclusion::Zuoguiwan can down-regulate the expressions of Ca2+ transport-associated proteins (Bone tissues) in rat osteoclasts, with an efficacy on osteoporosis.

Regulatory Effect of Guilu Bugu Prescription in Treating Th17/ Treg Cell Factors of Patients with Postmenopausal Osteoporosis Based on Syndrome Differentiation / 中国实验方剂学杂志

Objective:To observe the clinical efficacy Guilu Bugu prescription in treating postmenopausal osteoporosis （PMO） with deficiency of liver and kidney Yin based on syndrome differentiation and the effect on Th17/Treg cell factors. Method:One hundred and forty patients were randomly divided into observation group （70 cases） and control group （70 cases） by random number table. Both groups' patients got basic treatment of western medicine. Patients in control group got Jintiange capsules， 3 grains/time， 3 times/day. Patients in observation group got Guilu Bugu prescription， 1 dose/day. The treatment lasted for 6 months. And the 6-month follow-up was recorded. Before treatment， at the 6th month after treatment and at the 6th month of follow-up， bone density of lumbar vertebra L2-4 were detected by DXA， and Lumbar BMD were detected by QCT. Before treatment， at the 3rd and 6th month after treatment， deficiency of liver and kidney Yin and Chinese Osteoporosis-targeted quality of life questionnaire （COQOL） were scored. Before and after treatment， Estradiol （E2）， procollagen I amino terminal pro peptide （PINP）， osteoprotegerin （OPG）， collagen I cross linked C-terminal peptide （S-CTX）， tartrate resistant acid phosphatase （TRACP）， interleukin-17 （IL-17）， IL-22， IL-10， transforming growth factor-β1 （TGF-β1） were detected， and CD4+ cells， Th17 cells and Treg cells were calculated. And the safety was evaluated. Result:At the 6th month after treatment and the 6th month of follow-up， DXA （bone mineral density and T-value of lumbar L2-4） and QCT bone mineral density increased （P<0.01）， and the figures in observation group were all higher than those in control group （P<0.01）. At the 3rd and 6th month after treatment， scores of deficiency of liver and kidney Yin and quality of life were all lower than those in control group （P<0.01）. Levels of PINP， S-CTX， TRACP， Th17 cells， ratio of Th17 and Treg， IL-17 and IL-22 were all lower than those in control group （P<0.01）， and levels of OPG， E2， Treg， IL-10 and TGF-β1 were all higher than those in control group （P<0.01）.There was no adverse reaction related to Guilu Bugu prescription. Conclusion:Based on the supplementation of calcium and vitamin D， Guilu Bugu prescription can further improve the bone mineral density， raise the estrogen level， regulate the expressions of bone metabolism markers， Th17， Treg and relevant factors， reverse the imbalance of Th17/Treg， relieve the clinical symptoms and improve the quality of life， with a better efficacy than that of Jintange capsule and a high safety.

Effect of Recipes for Tonifying Kidney and Replenishing Qi on Learning and Memory-related Genes in Aged Rats / 中国实验方剂学杂志

Objective:To observe the effects of recipes for tonifying kidney and replenishing Qi, Zuoguiwan (ZG) and Yiqi Congming Tang(YQ) on memory capacity, expressions of learning and memory-related genes expression, and explore the changes in relevant epigenetic modification enzymes. Method:SD male rats with natural aging (24 months old) were used as animal models and randomly divided into aged control group, aged ZG group(12.12 g·kg-1), aged YQ group(10.18 g·kg-1), aged compound group(11.15 g·kg-1) and aged antagonist RU38486 group(5×10-3g·kg-1). Another 5 months old male SD rats were included as the young control group. Morris water maze method was used to observe the spatial learning and memory ability of the rats. The co-localizations of histone deacetylase 2 (HDAC2) and methyl CpG binding protein 2 (MeCP2) in hippocampus of rats in each group were observed by laser confocal microscope. The changes in expressions of glucocorticoid receptor (GR), synapsin1(Syn-1), HDAC2, and histone acetyltransferase 1(HAT1) proteins in hippocampus of each group were detected by Western blot, and mRNA expression of HDAC2 was detected by Real-time fluorescence quantitative polymerase Chain reaction (Real-time PCR). At the same time, the effects of ZG, YQ and compound decoction in alleviating the above-mentioned abnormal changes were observed. Result:Compared with the young control group(control group), the latency of the aged control group was significantly prolonged (PPPPPPPConclusion:ZG group, YQ group, and compound group can improve the spatial learning and memory abilities of aged rats by increasing the expression of learning-memory-associated protein GR and epigenetic modification enzyme HAT1, and reducing the expression of HDAC2 and the co-localization of HDAC2 protein and MeCP2 in the nucleus.

Review of Zuoguiwan on Animal Models and Evaluation in Prevention and Treatment of Osteoporosis / 中国实验方剂学杂志

Zuoguiwan is a classic prescription for replenishing vital essence, tonifying kidney-Yin and nourishing the bone marrow. Zuoguiwan is one of the effective prescriptions for the prevention and treatment of osteoporosis (OP), which reflects the thought of Reinforcing Yang from Yin. The OP animal model simulates the pathological state and pathogenesis of OP in human, which is an important means to research the pathogenesis of OP and verify the effect of drugs. In this paper, two kinds of animal models and characteristics of Zuoguiwan in the prevention and treatment of osteoporosis were discussed in details. They are the primary osteoporosis animal models, including ovariectomized animal models and spontaneous elderly osteoporosis animal models, and the secondary osteoporosis models, including glucocorticoid-induced animal models, cyclophosphamide-induced animal models and subtotal nephrectomy animal models. The evaluation methods of Zuoguiwan in preventing and treating OP, including bone absorption markers and bone formation markers analyzed by enzyme-linked immunosorbent assay (ELISA), bone mineral density detected with dual-energy X-ray, the number of trabeculae, trabecular segregation, trabecular thickness, bone volume/tissue volume ratio and bone surface/volume ratio analyzed using micro-CT, bone pathological morphology observed by hematoxylin-eosin staining, bone biomechanical properties, such as the maximum load force based on biomechanical test. In order to provide scientific reference for the basic and clinical research of Zuoguiwan, the OP animal models and the pharmacodynamic effect of Zuoguiwan are evaluated comprehensively with five different and objective evaluation methods. However, the animal model of OP needs to be further optimized to highlight the pathogenesis and syndrome characteristics of Zuoguiwan in the treatment of OP.

Effect of Zuoguiwan on mRNA Expression of Orexin and Its Receptors in Hypothalamus of Ovariectomized Rats / 中国实验方剂学杂志

Objective: To observe the mRNA levels of Orexin and its receptors in the hypothalamus of ovariectomized osteoporosis rats, in order to explore the pathogenesis of postmenopausal osteoporosis(PMOP) and the mechanism Zuoguiwan. Method: An osteoporosis model induced by ovariectomy was established in rats. Totally 32 female Sprague-Dawley (SD) rats were randomly divided into sham-operated group, ovariectomized model group, 17β-estradiol treated positive group, and Zuoguiwan group, with 8 rates in each group. After 12 weeks of intragastric administration, the bone mineral density (BMD) and trabecular microstructural changes of femur were detected by micro-CT (μ-CT), and the morphological changes of bone tissue were observed by hematoxylin-eosin staining (HE) staining. The markers of bone turnover in serum osteocalcin (OCN), N-terminal propeptide of type Ⅰ procollagen (PINP), tartrate-resistant acid phosphatase (TRAP) were measured using enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of orexin, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) were measured by Real-time PCR. Result: Compared with sham-operated group, the μ-CT showed that BMD, bone volume fraction(BV/TV), trabecular thickness(Tb. Th)and trabecular number(Tb. N)in ovariectomized model group were significantly decreased (PPN-terminal propeptide of type Ⅰ procollagen (PINP) levels decreased, whereas tartrate-resistant acid phosphatase (TRAP) content increased (PPPPPPPPPPConclusion: Decreased mRNA levels of Orexin and its receptors in the hypothalamus may be one of the mechanisms of PMOP. Zuoguiwan may correct the imbalance of bone metabolism, improve the trabecular microstructure and improve bone by up-regulating the mRNA expressions of Orexin and its receptors in the hypothalamus, density, so as to show a therapeutic effect on PMOP.

Effect of Zuoguiwan in Improving BMD and Trabecular Bone Microarchitecture in Ovariectomy-induced Osteoporosis Rats by Regulating RANKL/OPG Pathway Mediated by β2AR / 中国实验方剂学杂志

Objective: To investigate the mechanism of Zuoguiwan in treating ovariectomy-induced osteoporosis rats by receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) signaling pathway mediated by β2-adrenergic receptor (β2AR). Method: Forty Sprague-Dawley female rats were randomly divided into Sham-operated group (Sham) and four ovariectomized (OVX) subgroups. Rats in Sham and OVX groups were treated with 17β-estradiol (50 μg·kg-1·d-1), and low and high-dose ZGW (2.3,4.6 g·kg-1 lyophilized powder) for 3 months, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum markers of bone turnover. Micro-CT was used to evaluate and measure trabecular bone microarchitecture and bone mineral density (BMD) of the right distal femur. Western blot analysis and Real-time PCR were used to measure mRNA and protein expressions of β2AR, OPG and RANKL. Result: After 12 weeks of treatment with Zuoguiwan, the level of serum β-cross-linked c-telopeptide of type Ι collagen (β-CTX) (PPPβ2AR in the hypothalamus (PPConclusion: The mechanism of Zuoguiwan in alleviating BMD and trabecular bone microarchitecture in ovariectomy-induced osteoporosis rats might be related to the regulation of RANKL/OPG Pathway mediated by β2AR.

Intervention effects of Zuoguiwan containing serum on osteoblast through ERK1/2 and Wnt/β-catenin signaling pathway in models with kidney-Yang-deficiency, kidney-Yin-deficiency osteoporosis syndromes / 中国中药杂志

To clarify the effects of Zuoguiwan containing serum on osteoblast proliferation and alkaline phosphatase(ALP) expression and its effects on the expression of β-catenin, ERK1, ERK2 mRNA and protein of osteoblast through ERK1/2, Wnt/β-catenin signaling pathway in models with osteoporosis(OP) kidney-Yang-deficiency, osteoporosis(OP) kidney-Yin-deficiency syndrome. Rat osteoporosis models were established by ovariectomy surgery, and 10 weeks after surgery, hydrocortisone was injected and thyroxine was administered by intragastric administration to establish OP kidney-Yang-deficiency rat model, and OP kidney-Yin-deficiency rat model. Osteoblasts were obtained from 24 h newborn rat skull and were identified by alkaline phosphatase and alizarin red staining. Zuoguiwan containing serum of OP, OP kidney-Yang-deficiency, and OP kidney-Yin-deficiency, as well as the blank serum were used to intervene the osteoblast, and the cells proliferation was detected by MTS. ELISA assay was used to detect ALP expression. RT-PCR assay was used to detect the mRNA expression of ERK1, ERK2, β-catenin and protein expression levels were detected by Western blot. The results showed that Zuoguiwan containing serum in OP kidney-Yin-deficiency model had stronger effect than OP kidney-Yang-deficiency in promoting osteoblast proliferation, ALP expression, osteoblast ERK1/2, Wnt/β-catenin signaling pathway related factors β-catenin, ERK1, ERK2 mRNA and protein expression levels. This was consistent with the TCM theory of "Zuoguiwan nourishes kidney Yin", providing a scientific basis for the clinical and dialectical treatment of osteoporosis. Zuoguiwan could regulate the proliferation and differentiation of bone cells by ERK1/2 and Wnt/β-catenin signaling pathway, which may be one of the mechanisms of Zuoguiwan for the prevention of osteoporosis.

Effects of Zuoguiwan and Youguiwan on Immunohistochemical Expression of Lymphocyte Subpopulation in Central Nervous System of EAE Rats / 中国中医药信息杂志

Objective To observe the immunohistochemical expression of lymphocyte subpopulationin in brain and spinal cord tissue,and explore the mechanism of Zuogui wan and Yougui wan on rats with experimental autoimmune encephalomyelitis(EAE).Methods Lewis rats were immunized with the myelin basic protein(MBP).120 rats were grouping randomly into normal group,EAE group,prednisone group,Zuoguiwan group and Youguiwan group after post immunization(PI).Rats in normal group and EAE group were administered normal soline,each 3 mL/d.Rats in prednisone group were administered suspension of prednisone after developed clinical signs,each 5 mg/kg.Rats in Zuoguiwan group were administered suspension of Zuoguiwan,each 2 g/kg,and rats in Youguiwan group were administered suspension of Youguiwan,each 3 g/kg.On 15th and 27th day after PI,rats were killed and the immunohistochemical staining was performed on the sections of brain and spinal cord.Results On 15th and 27th day after PI,the expression of CD4+ in brain and spinal cord tissue of EAE group were significantly higher than that in normal group(P