Effect of Modified Huangqi Guizhi Wuwutang on Apoptosis-related Bax and Caspase-12 of Sciatic Nerve Cells in Diabetes Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of modified Huangqi Guizhi Wuwutang （MHGW） on the protein and mRNA expression of B-cell lymphoma-2-associated X protein （Bax） and cysteinyl aspartate specific proteinase-12 （Caspase-12） related to the apoptosis of sciatic nerve cells in diabetes rats to explore the mechanism of MHGW in the treatment of peripheral neuropathy in diabetes. MethodAnimal experiments were conducted. A diabetes model was induced in sixty male sprague-dawley （SD） rats by feeding on a high-sugar and high-fat diet combined with streptozotocin （STZ） intraperitoneal injection. Rats with random blood glucose levels ≥ 16.7 mmol·L-1 for three consecutive days were considered to have successfully developed diabetes. Forty-eight rats that successfully developed diabetes were randomly divided into a model group， an α-lipoic acid group （0.026 8 g·kg-1·d-1）， a high-dose MHGW group （2.5 g·kg-1·d-1）， and a low-dose MHGW group （1.25 g·kg-1·d-1）， with 12 rats in each group. Another 10 rats were assigned to the normal group. Body weight and random blood glucose levels of the rats were monitored. At the end of a 16-week intervention period， the sciatic nerve conduction velocity of the rats was measured using the Key point electromyography collection system. The protein and mRNA expression of Bax and Caspase-12 in the sciatic nerve cells was detected by Western blot analysis and real-time quantitative polymerase chain reaction （Real-time PCR）， respectively. ResultCompared with the normal group， the model group showed a significant decrease in body weight （P<0.01） and a significant increase in random blood glucose levels （P<0.01）. After a 16-week intervention， compared with the model group， the high-dose MHGW group exhibited a significant increase in body weight （P<0.05）， while there were no statistically significant differences in body weight changes among the other treatment groups. Random blood glucose levels significantly decreased in all treatment groups （P<0.01）. After 16 weeks of intervention， compared with the normal group， the model group had significantly reduced motor and sensory nerve conduction velocities （P<0.01）. Compared with the model group， all treatment groups showed significant increases in motor and sensory nerve conduction velocities （P<0.05， P<0.01）. The expression of Bax and Caspase-12 proteins in the sciatic nerve cells was significantly elevated in the model group compared with that in the normal group （P<0.01）. In contrast， all treatment groups showed significant reductions in the expression of Bax and Caspase-12 proteins in the sciatic nerve cells as compared with that in the model group （P<0.01）. The expression of Bax and Caspase-12 mRNA in the sciatic nerve cells significantly increased in the model group compared with that in the normal group （P<0.01）. Compared with the model group， the α-lipoic acid group and the high-dose MHGW group showed significant reductions in the expression of Bax mRNA in the sciatic nerve cells （P<0.05， P<0.01）， while the low-dose MHGW group showed a decreasing trend in the expression of Bax mRNA. The expression of Caspase-12 mRNA in the sciatic nerve cells significantly decreased in all treatment groups （P<0.01）. ConclusionMHGW may improve and repair sciatic nerve damage in diabetes rats by inhibiting sciatic nerve cell apoptosis.

Effect of Modified Huangqi Guizhi Wuwutang on Stress-related IRE1α/CHOP Pathway of Sciatic Nerve Endoplasmic Reticulum in Diabetes Rats / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of modified Huangqi Guizhi Wuwutang （MHGW） on endoplasmic reticulum stress in the sciatic nerve of diabetes rats based on the pathways of inositol-requiring enzyme 1α （IRE1α） and CCAAT/enhancer-binding protein homologous protein （CHOP）. MethodSixty rats were fed on a high-sugar and high-fat diet for six weeks， followed by intraperitoneal injection of streptozotocin at a dose of 35 mg·kg-1. Random blood glucose levels were measured three days later and rats with a sustained blood glucose level ≥ 16.7 mmol·L-1 were included in study （n=48）. The rats were randomly divided into a model group， an α-lipoic acid group （0.026 8 g·kg-1·d-1）， a high-dose MHGW group （2.5 g·kg-1·d-1）， and a low-dose MHGW group （1.25 g·kg-1·d-1）. Another 10 rats were assigned to the normal group. The intervention lasted for 16 weeks. After 16 weeks， the sciatic nerve structure of the rats in each group was observed under light microscopy using Luxol fast blue （LFB） staining. Transmission electron microscopy was used to observe the ultrastructure of the sciatic nerve. Chemiluminescence method was employed to measure the serum reactive oxygen species （ROS） levels. Western blot and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to evaluate the expression of p-IRE1α protein， IRE1α mRNA， CHOP protein， and CHOP mRNA in the sciatic nerve of the rats. ResultCompared with the normal group， the model group showed elevated serum ROS levels （P<0.01）. In contrast， the serum ROS levels were significantly reduced in the treatment groups compared with those in the model group （P<0.01）. The sciatic nerve of the model group showed pathological changes compared with that in the normal group， while the treatment groups exhibited improvement in sciatic nerve pathology compared with the model group. The protein expression of p-IRE1α and CHOP in the sciatic nerve significantly increased in the model group as compared with that in the normal group （P<0.01）. However， the treatment groups showed a significant decrease in the protein expression of p-IRE1α and CHOP in the sciatic nerve compared with the model group （P<0.05， P<0.01）. Furthermore， compared with the normal group， the model group showed upregulated mRNA expression of IRE1α and CHOP in the sciatic nerve （P<0.01）， while the treatment groups exhibited a significant decrease in the mRNA expression of IRE1α and CHOP compared with the model group （P<0.01）. ConclusionMHGW can alleviate endoplasmic reticulum stress-induced cell apoptosis and improve the structure and function of the sciatic nerve in diabetes rats by inhibiting the expression of IRE1α/CHOP pathway-related proteins and mRNA， thereby preventing and treating peripheral neuropathy in diabetes.

Effect of Huangqi Guizhi Wuwutang on Diabetic Peripheral Neuropathy in MKR Mice via Regulating Endoplasmic Reticulum Stress / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Huangqi Guizhi Wuwutang （HQGZWWT） in the treatment of diabetic peripheral neuropathy （DPN） in MKR mice via regulating endoplasmic reticulum （ER） stress. MethodThirty-two 8-week-old MKR mice （half were male and half were female） were fed with a high-fat diet for four weeks， and then 1% streptozotocin （STZ） was injected intraperitoneally for five days. After the blood glucose was stabilized， the mice were housed in the cage covered with ice bags for another one hour stimulation per day for four weeks. Mice with fasting blood glucose （FBG） value ≥11.1 mmol·L-1 were randomly divided into model group ， Huangqi Guizhi Wuwutang in original dosage group （30 g·kg-1·d-1）， Huangqi Guizhi Wuwutang in formula dosage group （6.25 g·kg-1·d-1）， and positive drug group （mecobalamin tablets， 0.17 mg·kg-1·d-1）. Another eight MKR mice of the same age were set as blank group and eight FVB mice were normal group. After four weeks of intragastric administration in each group， the change in FBG was tested， and hematoxylin and eosin （HE） staining and transmission electron microscope were used for observing the morphology of sciatic nerve tissue. In addition， the expression of c-Jun N-terminal kinase （JNK）， phosphorylated c-Jun N-terminal kinase （p-JNK） and inositol requiring enzyme 1α （IRE1α） proteins was determined by immunohistochemical test and Western blot （WB）. ResultCompared with the conditions in the normal group and blank group， the time of paw withdrawal， paw licking and tail flick in the model group was shortened （P<0.01）， and the conduction velocity of sciatic nerve was decreased （P<0.01）. Compared with the conditions in the model group， the behavioral and functional indicators were improved by HQGZWWT （P<0.05，P<0.01）. The immunohistochemical test revealed the JNK expression was elevated in the model group compared with the conditions in the normal group and blank group （P<0.05）， while that was lowered by HQGZWWT compared with the condition in the model group （P<0.05）. However， there was no difference among the treatment groups. According to the WB， the expression of IRE1α and p-JNK in the model group was enhanced compared with the conditions in the normal group and blank group （P<0.05，P<0.01）， while that was decreased by HQGZWWT compared with the condition in the model group （P<0.05，P<0.01）. No difference was observed between the HQGZWWTO and HQGZWWTF groups. ConclusionHQGZWWT can improve the neurophysiological function and pathological damage of sciatic nerve， which may be related to its delaying the ER stress response of sciatic nerve.

Chemical Composition Analysis and Characterization of Reference Sample of Huangqi Guizhi Wuwutang Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

ObjectiveTo analyze the chemical composition of the reference sample of Huangqi Guizhi Wuwutang （lyophilized powder）， and to provide quality markers for the formulation of quality standards of this formula. MethodUltra performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry （UPLC-Q-TOF-MS） was performed on a Waters ACQUITY UPLC™ HSS T3 column （2.1 mm×100 mm， 1.8 μm）， the mobile phase was methanol （A） -0.1% formic acid aqueous solution （B） for gradient elution （0-8 min， 1%-20%A； 8-10 min， 20%-30%A； 10-12 min， 30%-35%A； 12-14 min， 35%-40%A， 14-23 min， 40%-55%A， 23-27 min， 55%-99%A； 27-28 min， 99%A； 28-28.5 min， 99%-1%A； 28.5-30 min， 1%A）， the column temperature was 40 ℃， the injection volume was 2 μL， and the flow rate was 0.3 mL·min-1. The mass spectrometry data of the reference sample of Huangqi Guizhi Wuwutang （lyophilized powder） were collected under positive and negative ion modes. The conditions of mass spectrometry were electrospray ionization （ESI）， scanning range of m/z 50-1 200， and impact energy of 10-30 eV. UNIFI 1.8 and Progenesis QI 2.0 software were used to analyze and characterize the chemical constituents in reference sample of Huangqi Guizhi Wuwutang （lyophilized powder） combined with reference comparison and literature review. ResultA total of 123 chemical constituents were identified， including 33 flavonoids， 26 glycosides， 18 organic acids， 11 terpenoids， 7 phenylpropanoids， 4 gingerol， 3 alkaloids， 3 amino acids， 2 amides and 16 other compounds. ConclusionThe established method can quickly and accurately characterize the chemical components in the reference sample of Huangqi Guizhi Wuwutang （lyophilized powder）， which can provide a basis for the selection of quality evaluation indicators of this formula， and provide a reference for its preparation research.

Therapeutic Mechanism of Huangqi Guizhi Wuwutang on Rheumatoid Arthritis / 中国实验方剂学杂志

ObjectiveTo reveal the mechanism of action of Huangqi Guizhi Wuwutang in the treatment of rheumatoid arthritis by pharmacological research based on its clinical application. MethodThe collagen-induced arthritis （CIA） rat model was established by injecting bovine type Ⅱ collagen and Freund's adjuvant at the tail， and was treated with different concentrations of Huangqi Guizhi Wuwutang. The rats were randomly divided into blank group， model group， methotrexate （0.9 mg·kg-1） group， and Huangqi Guizhi Wuwutang low- and high-dose （5.13， 20.52 g·kg-1·d-1） groups， with continuous intragastric administration for 4 weeks. The degree of joint swelling， weight， degree of foot swelling and arthritis index score were determined and the pathological changes of ankle joints were detected by hematoxylin and eosin （HE） staining to observe the therapeutic effect of Huangqi Guizhi Wuwutang on rheumatoid arthritis. In addition， enzyme-linked immunosorbent assay （ELISA） and Western blot were used to measure the expression of interleukin 1β （IL-1β）， interleukin 6 （IL-6）， interleukin 10 （IL-10） and tumor necrosis factor-α （TNF-α） in serum and the expression of nuclear factor kappa-B （NF-κB） pathway related proteins in synovial tissue， respectively to clarify the molecular mechanism of Huangqi Guizhi Wuwutang in the treatment of rheumatoid arthritis. ResultCompared with the conditions in blank group， the body weight and IL-10 level were decreased （P<0.01）， and the degree of foot swelling and arthritis index score， the levels of IL-1β， IL-6 and TNF-α， and the expression of NF-κB pathway related proteins were increased （P<0.01，） in the model group， with impaired morphology and function of the ankle joint. Additionally， compared with the model group， Huangqi Guizhi Wuwutang low- and high-dose groups had increased body weight of rats and IL-10 level （P<0.01）， and reduced degree of foot swelling and arthritis index score （P<0.05， P<0.01）， levels of IL-1β， IL-6 and TNF-α （P<0.01） and expression of NF-κB pathway related proteins （P<0.05， P<0.01）， with improved function and morphology of the ankle joint. ConclusionHuangqi Guizhi Wuwutang can significantly alleviate joint inflammatory injury by down-regulating NF-κB pathway and reducing the inflammatory response in CIA rats.

Research Progress of Rheumatoid Arthritis Treatment with Huangqi Guizhi Wuwutang / 中国实验方剂学杂志

Rheumatoid arthritis （RA） is a common immune-mediated inflammatory autoimmunity disease. Due to its clinical characteristics， RA is classified into the category of "Bi syndrome" in traditional Chinese medicine. Huangqi Guizhi Wuwutang （HGW）， originated from Synopsis of Golden Chamber Jinkui Yaolue， is composed of Astragali Radix， Cinnamomi Ramulus， Paeonial actiflora， Zingiberis Rhizoma Recens， and Jujubae Fructus. It has been used for treatment of blood "Bi syndrome" in clinical practice， and nowadays， it is also widely used for treatment of RA or RA with wind-cold-dampness Bi syndrome or Qi and blood deficiency Bi syndrome. In 2018， HGW was collected in the Catalogue of Ancient Classical Prescriptions （the First Batch）. In order to provide insight for standardized clinical medication and lay a solid foundation for its further development， we herein reviewed literatures of clinical and animal studies on HGW treatment in RA. According to the basic pathogenesis of RA， HGW can be used for treatment of general RA， RA with wind-cold-dampness Bi syndrome or Qi and blood deficiency Bi syndrome alone or in combination with other drugs. The mechanisms of HGW were related to inhibition of inflammatory cytokines expression， inhibition of lipid peroxidation of synovium， and promotion of apoptosis. However， many issues in the current research still needed to be addressed， such as lack of standardization of drug compatibility and dosage， lack of standardization of clinical trial scheme，undefined drug interaction and unclear safety on the combination of Chinese medicine with chemistry drugs， lack of proper syndrome animal model， and limited study about molecular mechanisms. We propose that future research should focus on the standardization of the clinical trial scheme and RA syndrome of HGW， clarify the principle of drug compatibility and dosage， drug interaction and safety. In terms of mechanistic study， the research should focus on animal models of RA syndrome using multiple dimensions such as genetics， transcription， and metabolism.

Protective Effect of Huangqi Guizhi Wuwutang Combined with Shengmaiyin on Cardiac Function of Diabetic Cardiomyopathy / 中国实验方剂学杂志

Objective:To observe the protective effect of Huangqi Guizhi Wuwutang combined with Shengmaiyin on the heart function in patients with diabetic cardiomyopathy and explore its anti-myocardial fibrosis and anti-inflammatory effects. Method:The 96 patients were randomly divided into observation group （48 cases） and control group （48 cases）. Both groups were given comprehensive measures to control blood sugar， blood lipids， blood pressure and heart failure. Patients in control group took Tongmai Jiangtang capsule， 3 granules/time， 3 times/day. Patients in observation group took Huangqi Guizhi Wuwutang combined with modified Shengmaiyin， 1 dose/day. The treatment courses were three months in both groups. Left ventricular ejection fraction （LVEF）， early diastolic peak velocity E peak/late diastolic peak velocity A peak （E/A）， left ventricular end diastolic diameter （LVEDd） and cardiac output per stroke （SV） through echocardiography were recorded before and after therapy. Cardiac troponin-I （cTn I）， troponin T （cTn-T）， creatine kinase isoenzyme -MB （CK-MB）， lactate dehydrogenase （LDH）， transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁） before and after treatment ， matrix metalloproteinase-2 （MMP-2）， insulin-like growth factor-1 （IGF-1）， interleukin-6 （IL-6）， IL-1， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， N-terminal pro-B-type natriuretic peptide （NT-proBNP）， soluble ST2 （sST2） and galectin-3 （Gal-3） levels were detected. Symptom of cardiac insufficiency and traditional Chinese medicine （TCM） syndrome score were evaluated before and after treatment. Result:The LVEF and E/A data in observation group were higher than those in control group （<italic>P</italic><0.01）. The levels of cTn-I， cTn-T， LDH and CK-MB in the observation group were lower than those in the control group （<italic>P</italic><0.01）. After treatment， the levels of TGF-<italic>β</italic>₁， MMP-2， IGF-1， IL-6， IL-1， TNF-<italic>α</italic>， NT-proBNP， sST2 and Gal-3 in the observation group decreased and were lower than those in the control group （<italic>P</italic><0.01）. The clinical efficacy of the observation group was better than that of the control group （<italic>Z</italic>=1.974，<italic>P</italic><0.05）. Conclusion:On the basis of conventional intervention of western medicine， Huangqi Guizhi Wuwutang combined with modified Shengmaiyin has anti-inflammatory and anti-myocardial fibrosis effects， with inhibitory effect on myocardial remodeling， and can reduce myocardial tissue damage to improve ventricular diastolic function and protect heart function. With such high clinical efficacy， it is worthy of clinical use.

Effect of Huangqi Guizhi Wuwutang on AGEs/RAGE/NF-κB Signaling Pathway in Diabetic Peripheral Neuropathy Rats / 中国实验方剂学杂志

Objective:To study the effect of Huangqi Guizhi Wuwutang on receptor of advanced glycation end products(AGEs)/advanced glycation endproducts (RAGE)/nuclear transcription factor-kappa B p65 (NF-κB p65) signaling pathway in the diabetic peripheral neuropathy rats through an animal modeling experiment, and discuss the mechanism of Huangqi Guizhi Wuwutang in alleviating diabetic peripheral neuropathy. Method:Rat model of diabetic peripheral neuropathy was established by high-fat diet and intraperitoneal injection with streptozotocin (STZ). After successful modeling, Huangqi Guizhi Wuwutang intervention began in the fifth week. The patients in high-dose group (19.40 g∙kg-1∙d-1), middle-dose group (4.85 g∙kg-1∙d-1) and low-dose group (2.43 g∙kg-1∙d-1) were given by gavage continuously for 12 weeks. The western medicine control group was given 25 mg∙kg-1∙d-1 by gavage. After the experiment, serum interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect RAGE and NF-κB p65 mRNA expressions in sciatic nerve tissue. The expressions of RAGE, NF-κB and phosphorylation(p)-NF-κB p65 proteins in sciatic nerve tissues were detected by Western blot (WB). Result:Compared with the normal group, serum IL-1β and TNF-α protein levels, RAGE mRNA and NF-κB p65 mRNA levels, RAGE protein, NF-κB p65 protein and p-NF-κB p65 protein levels were significantly increased in the model group (P<0.01), the ratio of p-NF-κB p65 to NF-κB p65 was increased, and the phosphorylation of NF-κB p65 was enhanced (P<0.01). After the intervention of Huangqi Guizhi Wuwutang, compared with the model group, serum IL-1β and TNF-α protein levels, RAGE and NF-κB p65 mRNA levels, RAGE protein, NF-κB p65 protein and p-NF-κB p65 protein levels were all decreased (P<0.01), the ratio of p-NF-κB p65 to NF-κB p65 was decreased in high-dose group (P<0.01). The effect was obvious with the increase of dose of astragalus cassia twig. Conclusion:Huangqi Guizhi Wuwutang can alleviate diabetic peripheral neuropathy, and its mechanism may be related to blocking the expression of RAGE on tissue cell surface in AGEs/RAGE/NF-κB signaling pathway, inhibiting the activation of NF-κB and inducing TNF-α triggered oxidative stress and excessive inflammatory response, so as to avoid cell damage and dysfunction.

Clinical Efficacy of Jiwei Huangqi Guizhi Wuwutang Combined with Xingnao Kaiqiao Acupuncture on Shouder-hand Syndrome / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of Jiwei Huangqi Guizhi Wuwutang combined with Xingnao Kaiqiao acupuncture on shouder-hand syndrome (SHS), and its effect on neurogenic inflammatory factors and hemorheology. Method:One hundred and forty-eight patients were randomly divided into control group and observation group (74 cases). Both groups' patients got rehabilitation measures, such as diclofenac sodium sustained release tablets for two to four weeks, 75 min/time, 1 time/day, and patients with apparent swelling got prednisone acetate tablets for one to two weeks, 10 min/time, 1 time/day. And patients in control group got Xingnao Kaiqiao acupuncture, 1 time/day, 6 times/week. Control grouporal Naoxintong capsule 4 tablets/time,3 times/day, patients in observation group were added with Jiwei Huangqi Guizhi Wuwutang, 1 dose/day. The courses of treatment were 4 weeks. Before and after treatment, shoulder hand syndrome scale (SHSS), the upperextremities of the Fugl-meyer movement assessment (U-FMA), ability of daily life activities (ADL), Qi deficiency and blood stasis syndrome and clinical efficacy were scored, disappearing time of pain and swelling were recorded, and levels of calcitonin gene related peptide (CGRP), substance P (SP), bradykinin (BK) and hemorheology were detected. Result:The clinical efficacy in observation group was better than that in control group (Z=2.106, P<0.05). And scores of sensory, autonomic, motion according to SHSS scale and the total scale of SHSS were all lower than those in control group (P<0.01). Disappearing time of pain and swelling were shorten than those in control group (P<0.01). After treatment, scores of U-FMA and ADL were higher than those in control group (P<0.01), while score of syndrome of Qi deficiency and blood stasis was lower than that in control group (P<0.01). And level CGRP was higher than that in control group (P<0.01), and levels of SP and BK were lower than those in control group (P<0.01). After treatment, whole blood viscosity (high cut, low cut), plasma viscosity, fibrinogen and platelet aggregation rate were all lower than those in control group (P<0.05). Conclusion:In addition to the conventional western medicine therapy, Jiwei Huangqi Guizhi Wuwutang combined with Xingnao Kaiqiao acupuncture can reduce the severity of SHS and clinical syndromes of traditional Chinese medicine, shorten the course of disease, improve the motor function of upper limbs, inhibit the neurogenic inflammatory reaction, and improve the blood flow, the ability of daily life and the clinical efficacy of patients..