Steamed Panax notoginseng and its Saponins Inhibit the Migration and Induce the Apoptosis of Neutrophils in a Zebrafish Tail-Fin Amputation Model.

Panax notoginseng (PN) is a Chinese medicinal herb that is traditionally used to treat inflammation and immune-related diseases. Its major active constituents are saponins, the types and levels of which can be changed in the process of steaming. These differences in saponins are causally relevant to the differences in the therapeutic efficacies of raw and steamed PN. In this study, we have prepared the extracts of steamed PN (SPNE) with 70% ethanol and investigated their immunomodulatory effect using a zebrafish tail-fin amputation model. A fingerprint-effect relationship analysis was performed to uncover active constituents of SPNE samples related to the inhibitory effect on neutrophil number. The results showed that SPNE significantly inhibited the neutrophil number at the amputation site of zebrafish larvae. And SPNE extracts steamed at higher temperatures and for longer time periods showed a stronger inhibitory effect. Ginsenosides Rh1, Rk3, Rh4, 20(S)-Rg3, and 20(R)-Rg3, of which the levels were increased along with the duration of steaming, were found to be the major active constituents contributing to the neutrophil-inhibiting effect of SPNE. By additionally investigating the number of neutrophils in the entire tail of zebrafish larvae and performing TUNEL assays, we found that the decreased number of neutrophils at the amputation site was due to both the inhibition of their migration and apoptosis-inducing effects of the ginsenosides in SPNE on neutrophils. Among them, Rh1 and 20(R)-Rg3 did not affect the number of neutrophils at the entire tail, suggesting that they only inhibit the migration of neutrophils. In contrast, ginsenosides Rk3, Rh4, 20(S)-Rg3, and SPNE did not only inhibit the migration of neutrophils but also promoted neutrophilic cell death. In conclusion, this study sheds light on how SPNE, in particular the ginsenosides it contains, plays a role in immune modulation.

Market access for Chinese herbal medicinal products in Europe-A ten-year review of relevant products, policies, and challenges.

BACKGROUND: With increased consumer demand in Europe for natural and efficacious health products, the use of herbal products in the market is rising. Products of Chinese herbal medicine (CHM) could greatly expand European consumer options; however, only seven herbal medicinal products (HMPs) based on CHM formulae have been registered in the European Union (EU) since 2012. PURPOSE: This study reviews the ten-year registration status of HMPs based on CHM formulae in Europe and identifies major challenges and possible solutions for pharmaceutical companies seeking market access for new HMPs. METHODS: An overview of relevant EU regulations identifies pathways to market access in EU countries for CHM products. A discussion of successful attempts to register HMPs based on CHM formulae since 2012 highlights specific challenges that applicants can expect to face. RESULTS: CHM products can enter the EU market as HMPs through the full or well-established use marketing authorization, or through the simplified registration procedure. Alternatively, some CHM products have entered the market as dietary supplements, nutritional foods, and agricultural products; however, under these categories, claims for medicinal use cannot be advertised. Since the registration of the first CHM product, Diao Xin Xue Kang (with the single component of Dioscorea nipponica rhizome), in 2012, only six other HMPs based on CHM formulae have been successfully registered. Among these, four are mono-component products. The remaining two products contain combinations of several herbal ingredients. It is more difficult to register combination products than mono-component products, due to their more complex composition and differences in registration requirements (esp. concerning establishing indications) in China and Europe. CONCLUSIONS: To promote the successful registration of CHM products in Europe, pharmaceutical companies are advised to: demonstrate full control of, and the ability to test, their supply chain and manufacturing procedures following the guidance of European competent authorities; carefully adhere to all steps of the registration process and advices from European competent authorities; take the medication habits and pharmaceutical needs of European market into consideration; and establish collaboration with European local organizations, as appropriate.

Steamed Panax notoginseng attenuates renal anemia in an adenine-induced mouse model of chronic kidney disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng (PN) (Burk.) F. H. Chen is a medicinal herb used to treat blood disorders since ancient times, of which the steamed form exhibits the anti-anemia effect and acts with a "blood-tonifying" function according to the traditional use. However, its pharmacological effect and mechanism on alleviating renal anemia (RA) are still unclear. AIMS OF THE STUDY: The study aims to investigate the effect of steamed Panax notoginseng (SPN) to attenuate RA and its underlying mechanism based on the model of adenine-induced RA mice. MATERIALS AND METHODS: Seventy mice were randomly divided into seven groups of ten: the control group, model group, the erythropoietin (EPO) group, the Fufang E'jiao Jiang (FEJ) group, the high-dose steamed PN (H-SPN) group, the middle-dose steamed PN (M-SPN) group, and the low-dose steamed PN (L-SPN) group. The adenine induction RA model was applied to assess the "blood enriching" function of SPN. The blood routine indexes, erythrocyte fragility, pathologic morphology of kidney tissue and the expression levels of related cytokines and proteins in the mice were detected after 3-week administration with SPN and positive drugs. RESULTS: Our study provided evidences that SPN could ameliorate RA. Compared with the control group, SPN could attenuate RA by significantly increasing the numbers of peripheral blood cells (p < 0.01), improving the erythrocyte fragility (p < 0.01), and restoring the expression of EPO mRNA in the kidneys and EPO receptor mRNA in bone marrow nucleated cells. The expression of TGF-ß1 mRNA was declined and the expression of HGF mRNA was significantly increased in a dose-dependent way after the treatment of SPN. Additionally, the expression of Bcl-2 and Bcl-2/Bax ratio in the kidneys were significantly increased. In contrast, there was a highly significant decrease in the expression of Bax (p < 0.01), following SPN treatment. CONCLUSION: SPN could alleviate RA by promoting the overall hematopoiesis and inhibiting the progress of renal injury in mice.

[Aging-related change of erectile function and the expression of SIRT1].

OBJECTIVE: To explore aging-related changes in erectile function and the expressions of SIRT1 and other relevant factors in rats. METHODS: We divided 40 male SD rats into four age groups of equal number: 2-month-old (2 mo), 8-month-old (8 mo), 14-month-old (14 mo), and 20-month-old (20 mo), measured the intracavernous pressure (ICP), mean arterial pressure (MAP), and ICP/MAP ratio by electrostimulation of the cavernous nerve, evaluated fibrosis in the corpus cavernosum by Masson's trichrome staining, detected the expressions of SIRT1, P53, and FOXO3a by Western blot, and determined the levels of NO and cGMP using the NO/cGMP kit. RESULTS: Both the ICP/MAP ratio and the cGMP level were elevated with aging, reaching the peak at 8 months and then gradually decreased. Masson staining showed an aging-related increase of collagen fibers in the corpus cavernosum.The expression of SIRT1 was reduced while those of P53 and FOXO3a increased with aging. CONCLUSIONS: Aging-related erectile dysfunction may be attributed to the reduced activity of the NO/cGMP pathway, apoptosis and oxidative stress, and SIRT1 may play a role in aging-related erectile dysfunction.

[Combination of acupressure and magnetic sticker improved the quality of life in patients with advanced gastroenteric tumor: a clinical observation].

OBJECTIVE: To explore the clinical effect of combination of acupressure and magnetic sticker on the quality of life (QOL) including appetite, defecation, and sleep in patients with advanced gastroenteric tumor. METHODS: Totally 147 patients with advanced gastroenteric tumor were assigned to 4 groups according to different treatment methods, i.e., the supportive treatment group (A, 20 cases), the acupressure treatment group (B, 41 cases), the magnetic sticker treatment group (C, 40 cases), and a combination of acupressure and magnetic sticker treatment group (D, 46 cases). They were respectively treated with different methods, supportive treatment for group A, acupressure for group B, magnetic sticker for group C, and a combination of acupressure and magnetic sticker for group D. The scores of food intake, defecation frequency, sleep time, Karnofsky, and QOL were compared before treatment and at day 14 after treatment. RESULTS: After treatment, the scores of food intake, defecation frequency, and sleep time were obviously improved in B, C and D groups (P < 0.01). There was statistical difference between group D and group A (P < 0.01). In addition, in comparison with A group, both Karnofsky score and QOL score increased in B, C and D groups (P < 0.01). CONCLUSION: The assisted therapy of the combination of acupressure and magnetic sticker could ameliorate QOL such as the digestive functions and sleep in patients with advanced gastroenteric tumor.

Low-dose of multi-glycoside of Tripterygium wilfordii Hook. f., a natural regulator of TGF-ß1/Smad signaling activity improves adriamycin-induced glomerulosclerosis in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Transforming growth factor (TGF)-ß1/Smad signaling pathway plays a critical role in the prolonged glomerulosclerosis (GS), which is an important determinant during the progression in chronic kidney disease (CKD). For recent 30 years, multi-glycoside of Tripterygium wilfordii Hook. f. (GTW), an extract from Chinese herbal medicine has been proved clinically effective in improving GS in CKD in China. However, therapeutic mechanisms involved in vivo are still unclear. In this study, we aimed to explain the dose-effects and molecular mechanisms of GTW on GS by regulating TGF-ß1/Smad signaling activity in adriamycin (ADR)-induced nephropathy (ADRN). MATERIALS AND METHODS: Rats with ADRN, created by unilateral nephrectomy and twice adriamycin injections (ADR, 4 mg/kg and 2 mg/kg) within 4 weeks, were divided into four groups, the Sham group, the Vehicle group, the low-dose GTW-treated group, and the high-dose GTW-treated group, and that, sacrificed at the end of the 6th week after administration. Proteinuria, blood biochemical parameters, glomerulosclerotic morphological makers, podocyte shape, and nephrin expression were examined, respectively. Protein expressions of key signaling molecules in TGF-ß1/Smad pathway, such as TGF-ß1, Smad3, phosphorylated-Smad2/3 (p-Smad2/3), and Smad7, were also evaluated individually. RESULTS: The results indicated that the characterizations of ADRN involved the typical prolonged GS, a small amount of abnormal proteinuria, and the failing renal function; TGF-ß1/Smad signaling molecules, especially Smad3, p-Smad2/3, and Smad7 were activated in vivo, accompanied by the exasperation of glomerulosclerotic lesion; GTW at high-dose (100 mg/kg) and low-dose (50 mg/kg) could slightly ameliorate the prolonged GS and nephrin expression, furthermore, the anti-proliferative action of GTW at high-dose was superior to that at low-dose, but caused the significant liver injury; in ADRN model rats, protein expressions of TGF-ß1, p-Smad2/3, and Smad7 in the kidneys could be regulated with the treatment of GTW at low-dose. CONCLUSION: This study farther demonstrated that the low-dose of GTW, as a natural regulator in vivo, could effectively and safely ameliorate the prolonged GS in FSGS model, via the potential molecular mechanisms involving the reduction of ECM components and the suppression of TGF-ß1 over-expression, as well as the bidirectional regulation of TGF-ß1/Smad signaling activity.

Huangkui capsule, an extract from Abelmoschus manihot (L.) medic, ameliorates adriamycin-induced renal inflammation and glomerular injury via inhibiting p38MAPK signaling pathway activity in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Abelmoschus manihot (L.) medic (AM) is a natural medicinal plant used for the treatment of inflammatory diseases in China. Huangkui capsule (HKC), an extract from AM, has been proved clinically effective in improving renal inflammation and glomerular injury in chronic kidney disease (CKD). However, the dose-effects and the mechanisms involved in vivo are still unclear. AIM OF THE STUDY: This study was performed to examine the dose-effects of HKC on renal inflammation and glomerular lesion in adriamycin-induced nephropathy (ADRN), then to clarify the mechanisms in vivo of HKC by investigating its actions on modulating the activation of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway. MATERIALS AND METHODS: The rats with chronic ADRN, created by the unilateral nephrectomy and twice adriamycin injections (ADR, 4 mg/kg and 2mg/kg) within 4 weeks, were divided into four groups, a Sham group, a Vehicle group, a high-dose HKC group, and a low-dose HKC group, and that, sacrificed at the end of the 4th week after the administration. The rat's general status, renal morphological appearance, proteinuria, blood biochemical parameters, glomerular morphological changes, podocyte shape, and macrophage (ED1(+) and ED3(+) cells) infiltration in glomeruli were examined, respectively. The protein expressions of inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-2, as well as p38MAPK signaling molecules such as transforming growth factor (TGF)-ß1, p38MAPK, and phosphorylated-p38MAPK (p-p38MAPK), were also evaluated individually. RESULTS: HKC at high dose of 2g/kg/d not only significantly ameliorated the rat's general status, renal morphological appearance, proteinuria, albumin, and glomerulosclerosis, but also obviously reduced the infiltrated ED1(+) and ED3(+) macrophages in glomeruli and TNF-α protein expression in the kidney, in addition to these, evidently down-regulated TGF-ß1 and p-p38MAPK protein expressions in ADRN rats, but had no influence on podocyte shape and renal function. CONCLUSION: HKC could dose-dependently ameliorate renal inflammation and glomerular injury in ADRN rats, by way of reducing the infiltration and the activation of macrophages in glomeruli, and TNF-α protein expression in the kidney, as well as inhibiting p38MAPK signaling pathway activity via the down-regulation of p-p38MAPK and TGF-ß1 protein expressions in vivo.

Multi-glycoside of Tripterygium wilfordii Hook. f. reduces proteinuria through improving podocyte slit diaphragm dysfunction in anti-Thy1.1 glomerulonephritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) has been proved clinically effective in reducing proteinuria in chronic kidney disease in China. However, the mechanisms involved are still unclear. In this study we examined the effects of GTW at the different dosages on proteinuria and podocyte slit diaphragm (SD) dysfunction in anti-Thy1.1 glomerulonephritis (GN). MATERIALS AND METHODS: Rats with anti-Thy1.1 GN were divided into 2 groups, a GTW group and a vehicle group, and sacrificed at 30 min, on day 7, and on day 14 in Experiments 1, 2 and 3, respectively. The administration of GTW at the moderate and high doses was started 3 days before or at the same time of antibody injection till sacrifice. Proteinuria was determined in Experiments 1, 2, and 3. After sacrifice, the staining intensity of SD-associated key functional molecules including nephrin and podocin, podocyte structure, mesangial change, macrophage infiltration, and blood biochemical parameters were examined, respectively. Protein and mRNA expressions of nephrin and podocin in glomeruli were also investigated. Besides, liver histological characteristics were analyzed. RESULTS: In Experiment 1, GTW pretreatment at the medium dose (75 mg/kg body weight) caused no influence on the induction of anti-Thy1.1 GN and the basal nephrin expression. In Experiment 2, the high dosage (100mg/kg body weight) of GTW ameliorated proteinuria, the distribution of nephrin and podocin, mesangial proliferation, and the activated macrophage accumulation, as compared with vehicle group (P<0.05). Additionally, it increased mRNA and protein expressions of nephrin and podocin in glomeruli on day 7, but had no influence on podocyte structure. In Experiment 3, the medium dosage (75 mg/kg body weight) of GTW improved proteinuria, the partial matrix expansion, and the distribution of nephrin and podocin on day 14, as compared with anti-Thy1.1 GN rats (P<0.05). GTW at the high or moderate dose did not affect hepatic function on day 7 and on day 14. CONCLUSIONS: Podocyte SD dysfunction, such as the disordered distribution and down-regulation of nephrin and podocin expression, is critically involved in the pathogenesis of anti-Thy1.1 GN induced by mAb 1-22-3. The restoration of the distribution and expression of nephrin and podocin by GTW could be an important mechanism by which GTW ameliorates proteinuria and podocyte SD dysfunction.

[Preventive effects of multi-glycoside of Tripterygium wilfordii on glomerular lesions in experimental diabetic nephropathy].

OBJECTIVE: To observe the preventive effects of multi-glycoside of Tripterygium wilfordii (GTW) on glomerular lesions in experimental diabetic nephropathy (DN). METHOD: The DN model of rats was established with streptozotocin (STZ) and intervened with GTW. In the same time, normal, benazepril, and vehicle control groups were set up. After 8 weeks of oral treatment with GTW (50 mg x kg(-1) BW), benazepril (6 mg x kg(-1) BW), and vehicle (physiological saline), the changes of body weight, urine albumin (UA1b), blood glucose (BG), serum creatinine (Scr), blood urea nitrogen (BUN) and glomerular morphology were examined. In addition, the level of protein expression of alpha-smooth muscle actin (alpha-SMA) and collagen type I in glomeruli was determined by immunofluorescence. RESULT: Both GTW and benazepril reduced UA1b. GTW ameliorated glomerular injury, such as mesangial cell proliferation, alpha-SMA and collagen type I over-expression, in DN model. Compared with benazepril, beneficial effects of GTW on glomerulusclerosis were more significant (total cell number: GTW group 54.44 +/- 2.41, benazepril group microg/67.83 +/- 4.41, P < 0.05; alpha-SMA score: GTW group 1.98 +/- 0.52, benazepril group 2.27 +/- 0.46, P < 0.05; collagen type I score: GTW group 2.11 +/- 0.37, benazepril group 2.88 +/- 0.58, P < 0.05). CONCLUSION: Preventive effects of GTW on glomerular lesion in DN model are related to decreasing UA1b and ameliorating glomerulusclerosis.

[Suppressive effects of GTW treatment on infiltration of inflammatory cell in glomeruli in anti-Thy1.1 glomerulonephritis].

OBJECTIVE: To examine inhibition action of multi-glycoside of Tripterygium wilfordii (GTW) on infiltration of inflammatory cell in glomeruli with anti-Thy1.1 glomerulonephritis (anti-Thy1.1 GN), and to clarify its effects on inflammatory in vitro. METHOD: Two types of anti-Thy1.1 GN were induced in rats by a single or two intravenous injections with 500 microg of anti-Thy1.1 mAb 1-22-3. Rats were randomly divided into two groups, the GTW group and control group, and sacrificed on day 7 or on day 42 after induction of anti-Thy1.1 GN. Daily oral administration of different dose of GTW and distilled water as a control was started from 3 days before injection or at the same time of injection till the day of sacrifice. Proteinuria was determined during days 7 or during days 42. Infiltration of macrophage and T lymphocyte in glomeruli and mRNA expression of interleukin (IL)-2 and interferon (IFN)-gamma in renal tissue were examined. RESULT: Increase of infiltration of macrophage in reversible anti-Thy1.1 GN model, glomerular macrophage infiltration and IL-2 mRNA expansion were attenuated by higher dose of GTW (75 mg x kg(-1) x d(-1)), and increased accumulation of activated macrophage and T lymphocyte in irreversible anti-Thy1.1 GN model, accumulation of macrophage and T lymphocyte in glomeruli and mRNA expansion of IL-2 and IFN-gamma were decreased by middling dose of GTW (50 mg x kg(-1) x d(-1)) as well. Proteinuria was significantly ameliorated after GTW administration. CONCLUSION: The findings suggested that different dose of GTW can ameliorate infiltration of inflammatory cell in glomeruli with anti-Thy1.1 glomerulonephritis in vitro by decreasing the expression of IL-2 and IFN-gamma.