Effect and mechanism of Dahuang Zhechong Pills in improving liver aging in rats by regulating ROS-mediated PI3K/Akt/FoxO4 signaling pathway / 中国中药杂志

Recent studies have shown that the occurrence and development of common liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and liver cancer, are related to liver aging(LA). Therefore, to explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a traditional classic prescription in improving LA with multiple targets, the present study randomly divided 24 rats into a normal group, a model group, a DHZCP group, and a vitamin E(VE) group, with six rats in each group. The LA model was induced by continuous intraperitoneal injection of D-galactose(D-gal) in rats. For the LA model rats, the general situation was evaluated by aging phenotype and body weight(BW). LA was assessed by the pathological characteristics of hepatocyte senescence, hepatic function indexes, the staining characteristics of phosphorylated histone family 2A variant(γ-H2AX), and the expression levels of cell cycle arrest proteins(P21, P53, P16) and senescence-associated secretory phenotype(SASP) in the liver. The activation of the reactive oxygen species(ROS)-mediated phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/forkhead box protein O4(FoxO4) signaling pathway was estimated by hepatic ROS expression feature and the protein expression levels of the key signaling molecules in the PI3K/Akt/FoxO4 signaling pathway. The results showed that after the treatment with DHZCP or VE for 12 weeks, for the DHZCP and VE groups, the characterized aging phenotype, BW, pathological characteristics of hepatocyte senescence, hepatic function indexes, relative expression of ROS in the liver, protein expression levels of key signaling molecules including p-PI3K, p-Akt, and FoxO4 in the liver, staining characteristics of γ-H2AX, and the protein expression levels of P16, P21, P53, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the liver were improved, and the effects of DHZCP and VE were similar. Based on the D-gal-induced LA model in rats, this study demonstrates that DHZCP can ameliorate LA with multiple targets in vivo, and its effects and mechanism are related to regulating the activation of the ROS-mediated PI3K/Akt/FoxO4 signaling pathway in the liver. These findings are expected to provide new pharmacological evidence for the treatment of DHZCP in aging-related liver diseases.

Clinical Observation of Yishen Tongluo Decoction in Treating Chronic Glomerulonephritis with Spleen and Kidney Qi Deficiency and Blood Stasis Syndrome / 中国实验方剂学杂志

Objective:To observe the clinical efficacy as well as anti-inflammatory and anti-fibrosis mechanism of Yishen Tongluo decoction in the treatment of chronic glomerulonephritis （CGN） with spleen and kidney Qi deficiency and blood stasis syndrome. Method:According to the random number table method， one hundred and twenty patients were divided into control group （60 cases） and observation group （60 cases）. The two groups were given oral valsartan capsules， 160 mg/time， 1 time/day， and dipyridamole tablets orally， 50 mg/time， 3 times/day. Patients in control group additionally took Wubi Shanyao pills orally， 9 g/time， 2 times/day， while patients in observation group additionally took Yishen Tongluo decoction orally， 1 dose/day. Both groups were treated continuously for four months. Before and after treatment， 24 h urine total protein （24 h UTP）， creatinine （SCr）， urea nitrogen （BUN）， serum cystatin C （CysC）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-6 （IL-6）， IL-17， interferon-<italic>γ</italic> （IFN-<italic>γ</italic>）， transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁）， connective tissue growth factor （CTGF）， matrix metalloproteinase-9 （MMP-9）， tissue inhibitor of metalloproteinases （TMP-1） and hypoxia inducible factor-1<italic>α </italic>（HIF-1<italic>α</italic>） levels were detected in both groups. The scores of spleen and kidney Qi deficiency and blood stasis syndrome were graded. Urine protein， and urine red blood cells （urinary RBC） were monitored. Result:The 24 h UTP， SCr， BUN and CysC levels of the observation group were lower than those of the control group （<italic>P</italic><0.01）. The score of spleen and kidney Qi deficiency and blood stasis syndrome in the observation group was lower than that in the control group （<italic>P</italic><0.01）. The levels of TNF-<italic>α</italic>， IL-6， IL-17 and IFN-<italic>γ </italic>in the observation group were lower than those in the control group （<italic>P</italic><0.01）. The levels of TGF-<italic>β</italic>₁， CTGF， TMP-1 and HIF-1<italic>α</italic> in the observation group were lower than those in the control group （<italic>P</italic><0.01）. While MMP-9 level was higher than that in control group （<italic>P</italic><0.01）. The clinical effective rate was 88.33% （53/60） in the observation group， higher than 73.33% （44/60） in the control group （<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M2"><mml:msup><mml:mrow><mml:mi>χ</mml:mi></mml:mrow><mml:mrow><mml:mn mathvariant="normal">2</mml:mn></mml:mrow></mml:msup></mml:math><graphic specific-use="big" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="alternativeImage/155A9E27-C0C9-44b4-96FF-AF947372054E-M002.jpg"><?fx-imagestate width="3.30200005" height="3.64066648"?></graphic><graphic specific-use="small" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="alternativeImage/155A9E27-C0C9-44b4-96FF-AF947372054E-M002c.jpg"><?fx-imagestate width="3.30200005" height="3.64066648"?></graphic></alternatives></inline-formula>=4.356， <italic>P</italic><0.05）. Conclusion:On the basis of conventional Western medicine treatment， Yishen Tongluo decoction in the treatment of CGN patients with spleen and kidney Qi deficiency and blood stasis syndrome can reduce proteinuria， and improve traditional Chinese medicine （TCM） syndromes， with anti-inflammatory and anti-kidney fibrosis effects. Thereby， it plays a role in protecting renal function and delaying the malignant progression of renal function， with high clinical efficacy and value of clinical use.

Content Determination of 17 Quality Markers in Dahuang Zhechong Pills / 中国药房

OBJECTIVE：To establish the method for the content de termination of 17 quality markers in Dahuang zhechong pills（DHZCP）. METHODS ：HPLC method was adopted to determine the contents of 17 quality markers in 10 batches of DHZCP ， such as allantoin ，hypoxanthine，salidroside，hydroxypaeoniflorin，glycyrrhizin，isoglycyrrhizin，baicalin，p-methoxyphenylacetic acid，wogonin，cinnamic acid ，apigenin，naringin，norwogonin，aloe emodin ，rhein，chrysin，emodin. The determination was performed on Kromasil 100-5-C18（250 mm × 4.6 mm，5 μm）column with mobile phase consisted of 0.1％ phosphoric acid solution-acetonitrile （gradient elution ） at the flow rate of 1.0 mL/min. The column temperature was 30 ℃ ，the detection wavelength was 210 nm and the sample size was 20 μ L. RESULTS：The linear range of above 17 quality markers were 5.74-183.53，6.51-208.24，4.30-137.65，4.60-147.06，4.12-131.76，4.25-135.88，6.31-201.76，4.60-147.06，1.94-62.06，4.47- 142.94，0.69-22.06，2.29-73.24，2.33-74.41，1.42-45.29，6.65-212.94，1.11-35.44 and 1.47-47.06 μg/mL，respectively（all R2≥ 0.999 0）. RSDs of precision ，repeatability，stability and durability tests were all less than 2％（n＝6）；average recovery of 17 quality markers ranged from 96.31％ to 101.73％，and the RSDs were less than 3％（n＝6）. CONCLUSIONS ：The method is simple， rapid，speific，specise，reproducible，stable，accurate and durable ，and can be used for improving the quality standard of DHZCP.

Effect of Dahuang Zhechong Pills combined with TACE on VEGF, MMP-2, TGF-β1 and immune function of patients with primary liver cancer (blood stasis and collaterals blocking type) / 中国中药杂志

To investigate the effects of Dahuang Zhechong Pills combined with hepatic arterial chemoembolization(TACE) on tumor index and immune function of patients with primary liver cancer(blood stasis and collaterals blocking type), observe its application values in treatment of such patients, and provide effective treatment means for this disease. From June 2019 to December 2019, 79 patients with confirmed primary liver cancer(blood stasis and collaterals blocking type) treated in Wenzhou Hospital of Traditional Chinese Medicine were included in this study, all of which were grouped with random number table method before inclusion in this study. 40 patients in the control group were treated with TACE, while 39 patients in the observation group were treated with Dahuang Zhechong Pills combined with TACE. The efficacy was compared between two groups after 4 weeks of treatment. The immune function indexes of serum CD4~+ cells, CD4~+/CD8~+, CD3~+ cells of the observation group were higher than those in control group after treatment(P<0.05), and tumor indexes such as serum alpha-fetoprotein(AFP), carbohydrate antigen 199(CA199) and glutamic-pyruvic transaminase(ALT), total bilirubin(TBiL) levels were lower than those in the control group, with statistically significant differences(P<0.05). Plasma vascular endothelial growth factor(VEGF), transforming growth factor-β1(TGF-β1), and matrix metalloprotei-nase-2(MMP-2) levels in the observation group were lower than those in the control group after treatment, with statistically significant differences(P<0.05). The total effective rate of the observation group was 87.18%, higher than 67.50% in the control group, and the benefit rate was 94.87% in the observation group, higher than 85.00% in the control group(P<0.05). The total incidence of adverse reactions such as bone marrow suppression, gastrointestinal reaction, fever, renal function injury and peripheral nerve injury in the observation group was 48.72%, lower than 82.50% in the control group, with statistically significant difference(P<0.05). In summary, the combination of Dahuang Zhechong Pills with TACE could improve immunity, protect liver function, and reduce the risk of metastasis and the incidence of adverse reactions from chemotherapy, so it is worth popularizing for patients with primary liver cancer(blood stasis and collaterals blocking type).

Simultaneous Determination of 6 Kinds of Constituent in Dahuang Zhechong Pills by HPLC-PDA / 中国药房

OBJECTIVE： To establish a method for amygdalin， paeoniforin， baicalin， glycyrrhizic acid， emodin and chrysophanol in Dahuang zhechong pills. METHODS： HPLC method was adopted. The determination was performed on Welchrom C18 with mobile phase consisted of methanol-0.1％ phosphoric acid （gradient elution） at the flow rate of 1.0 mL/min. The column temperature was set at 30 ℃， and the detection wavelengths were set at 210 nm （amygdalin）， 230 nm （paeoniforin， baicalin） and 250 nm [glycyrrhizic acid （calculated with ammonium glycyrrhizinate）， emodin， chrysophanol]. The sample size was 10 μL. RESULTS： The linear ranges of amygdalin，paeonifiorin，baicalin，ammonium glycyrrhizinate， emodin and chrysophanol fell within the ranges of 21.028-157.71，2.052-90.390， 34.288-257.16， 8.252 0-61.890， 3.272 0-24.540，4.768 0-35.760 μg/mL （all r≥0.999 2）. The limits of detection were 0.105 0， 0.121 0， 0.068 6， 0.082 5， 0.024 6， 0.017 9 μg/mL. The limits of quantitation were     0.263 0， 0.362 0， 0.171 0， 0.268 0， 0.065 5， 0.047 7 μg/mL. RSDs of precision， stability（12 h）and reproducibility tests were lower than 2.00％ （n＝6）. The  average  recoveries were 96.01％-100.76％ （RSD＝1.63％，n＝6）， 97.09％-101.86％ （RSD＝1.50％，n＝6）， 99.70％-101.99％（RSD＝0.82％，n＝6），96.29％-99.52％（RSD＝1.35％，n＝6）， 98.47％-102.14％ （RSD＝1.35％， n＝6） and 97.19％-99.16％（RSD＝0.69％，n＝6），respectively. CONCLUSIONS： The method is accurate， simple and reproducible for simultaneous determination of amygdalin， paeoniforin， baicalin， glycyrrhizic acid， emodin and chrysophanol in Dahuang zhechong pills.

Influence of Dahuang Zhechong Pills Combined with Compound Gosspol Acetate Tablets on Serum Markers of Endometriosis Patiens After Operation with Laparoscopy / 中国中医药信息杂志

Objective To explore the clinical efficacy of Dahuang Zhechong Pills combined with compound gosspol acetate tablets for endometriosis (EM) after operation with laparoscopy; To observe changes in serum markers. Methods Totally 98 EM patients after operation with laparoscopy were divided into the control group (49 cases) and observation group (49 cases). All patients had laparoscopic conventional anti-infective, anti-inflammatory, nutritional support, maintenance of electrolyte and acid-base balance, for 14 d. The control group treated with compound gosspol acetate tablets, one tablet each time, once a day, orally after dinner; the observation group was given Dahuang Zhechong Pills on the basis of control group, two pills per time, twice a day, orally. The treatment course was 6 months. The clinical efficacy and symptom scores were evaluated in the two groups. The levels of MMP-3, CA-125, EMAb, IgG, FSH, E2 and LH were detected before and after treatment in the two groups. The adverse reaction was observed during the treatment. Results After the treatment, the total effective rate was 87.7％(43/49) in observation group, and 73.4％ (36/49) in control group, with statistical significance (P<0.05). After the treatment, the symptom scores were significantly lower in both groups, and the observation group was lower than the control group (P<0.05). After the treatment, the levels of MMP-3, CA-125, EMAb, FSH, E2 and LH decreased in both groups (P<0.05). The levels of MMP-3, CA-125, EMAb, FSH, E2 and LH in the observation group were lower than the control group (P<0.05). There were no serious adverse reactions in the two groups during the treatment period. Conclusion Dahuang Zhechong Pills combined with compound gosspol acetate tablets in the treatment of EM after operation with laparoscopy has a good clinical curative effect and can decrease the levels of MMP-3, CA-125, EMAb and estrogen levels.

Protective effects of Dahuang Zhechong Pills on mice with alcohol-induced liver fibrosis / 中成药

AIM To investigate the protective effects of Dahuang Zhechong (DHZC) Pills (Rhei Radix et Rhizoma,Eupolyphaga seu Steleophaga,Hirudo,etc.) against alcoholic liver fibrosis (ALF) injury in mice and to explore the underlying mechanisms.METHODS C57BL/6 male mice were used to build up ALF injury model,intervened with DHZC Pills.The serum of mice was examined for changes in alanine transaminase (ALT),aspartate aminotransferase (AST),interleukin-6 (IL-6),interleukin-10 (IL-10),interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α).Simultaneously,the deposit of collagen 1 (COL-1) and apoptotic cell death in liver tissues were analyzed by immunofluorescent and TUNEL assay,respectively.The expressions of cleaved caspase-3 (CC3) in livers were measured by Western blot.RESULTS Compared with the model group,the levels of serum ALT,AST,IL-6,IFN-γand TNF-α of mice in DHZC group were decreased significantly.And the level of serum IL-10 of mice in DHZC group was increased significantly.Mice in DHZC group had higher rates of COL-1 deposition and apoptotic cell death in liver tissues than those in the model group.Mice treated with DHZC Pills showed lower expression of CC3.CONCLUSION DHZC Pills confers protection against ALF injury in mice by inhibiting the generation of COL-1 and down-regulating apoptosis of liver cells death as a result of adjusting the levels of inflammatory factors.

Expression of Vascular Endothelial Growth Factor (VEGF) in Chronic Myelogenous Leukemia (CML) Patients and Influence of Serum Containing Dahuang Zhechong Pills on VEGF Expression in K562 Cells / 广州中医药大学学报

Objective To observe the effect of serum containing Dahuang Zhechong Pills on vascular endothelial growth factor ( VEGF) expression in K562 cells of chronic myelogenous leukemia ( CML) and to develop new antiangiogenic drugs for CML treatment. Methods Enzyme-linked immunosorbent assay ( ELISA) was used for the detection of serum VEGF content in 38 CML patients, 11 acute myelogenous leukemia patients and 9 healthy volunteers. SD rats were used for the preparation of serum containing Dahuang Zhechong Pills, and then ELISA was applied for the detection of VEGF content in K562 cells pretreated with serum containing Dahuang Zhechong Pills. Results Compared with the healthy volunteers, myelogenous leukemia patients at chronic phase and acute phase had high serum VEGF content ( P<0.05 or P<0.01) . Compared with blank serum control group, the secretion of VEGF in K562 cells pretreated with serum containing Dahuang Zhechong Pills was decreased obviously ( P<0.01) , and VEGF expression decreased with the increase of drug concentration. Conclusion VEGF content is increased in myelogenous leukemia patients at chronic phase and acute phase, which may be related to the pathogenesis, progress and prognosis of chronic myelogenous leukemia. Serum containing Dahuang Zhechong Pills can inhibit the expression of VEGF in chronic myelogenous leukemia K562 cells and decrease VEGF protein expression in concentration-dependent manner.

Effects of Dahuang Zhechong Pills on CD44,VEGF and the metastasis suppressor in mice with Lewis Lung carcinoma / 中华中医药杂志

Objective:By observing effects of Dahuang Zhechong Pills on CD44,VEGF and the metastasis suppressor in mice with Lewis Lung carcinoma,to discuss its mechanism of anti-tumor metastasis preliminarily.Methods:The mice were divided randomly into five groups the day after modeling and each group included ten mice.According to the pre-test,high-dose,medium-dose and low-dose were quantitated with 0.18g/ml,0.09 g/ml,0.045g/ml.The model group was intragastric administrated with NS 0.2ml for ten days;the chemotherapy group was Intraperitoneal administrated 50mg/kg CTX on the first day.Observing and numerating the pulmonary metastases with nodules by pulmonary pressure slide film on the 12th day.Based on immunohistochemical method to detect the expression of VEGF and the activity detection by flow cytometry CD44,Its anti-tumor mechanism and the role of target were preliminarily studied.Results:The inhibition rates of lung spontaneous metastasis in experimental groups of high(H),medium(M),low(L)dosages and the chemotherapy group were respectively 57.44 percent, 55.32 percent,38.72 percent,60.00 percent.CD44,respectively,values were 0.59?0.86,0.21?0.28,0.33?0.31,1.36?0.52; the experimental group(M)compared with model group,VEGF expression was significantly reduced,there was a significant difference(P0.05).Conclusion:Dahuang Zhechong Pills reduced the lung metastases incidence of C57BL mice with Lewis lung carcinoma and prolonged their lives.These results were obvious in the medium-dose experimental group.Medium-dose of Dahuang Zhechong Pills had effects on C57BL mice with Lewis lung carcinoma,whose CD44 activity were inhibited and the expression of VEGF was reduced.

Determination of rhien,emodin and chrysophanol in Dahuang Zhechong Pills by HPLC / 中成药

AIM To develop a method to determine rhien,emodin and chrysophanol contents in Dahuang Zhechong Pills (Radix et Rhizoma Rhei Preparata,Eupolyphaga seu Steleophaga,Hirudo,etc.). METHODS HPLC was used to determine rhien,emodin and chrysophanol contents in Dahuang Zhechong Pills.The separation was performed on SYMMETRY ○ column with methol-phosphate acid(0.1 % ) (6∶ 1) as a mobile phase and wavelength of UV detector was at 254 nm. RESULTS The resolution and the linearity of the method were good with the avevage recovery of rhien:101.92, RSD was 2.01 % ;emodin:97.74 % , RSD was 0.78 % ;chrysophanol, 98.66 % , RSD was 2.41 % ;respectively. CONCLUSION The method is simple and rapid and with satisfactoty results.It can be suitable for quality control of Dahuang Zhechong Pills.