Efficacy and safety of naotaifang capsules for hypertensive cerebral small vessel disease: Study protocol for a multicenter, randomized, double-blind, placebo-controlled clinical trial.

Background: Hypertensive cerebral small vessel disease (HT-CSVD) is a cerebrovascular clinical, imaging and pathological syndrome caused by hypertension (HT). The condition manifests with lesions in various vessels including intracranial small/arterioles, capillaries, and small/venules. Hypertensive cerebral small vessel disease has complex and diverse clinical manifestations. For instance, it can present as an acute stroke which progresses to cause cognitive decline, affective disorder, unstable gait, dysphagia, or abnormal urination. Moreover, hypertensive cerebral small vessel disease causes 25-30% of all cases of ischemic strokes and more than 50% of all cases of single or mixed dementias. The 1-year recurrence rate of stroke in cerebral small vessel disease patients with hypertension is 14%. In the early stage of development, the symptoms of hypertensive cerebral small vessel disease are concealed and often ignored by patients and even clinicians. Patients with an advanced hypertensive cerebral small vessel disease manifest with severe physical and mental dysfunction. Therefore, this condition has a substantial economic burden on affected families and society. Naotaifang (NTF) is potentially effective in improving microcirculation and neurofunction in patients with ischemic stroke. In this regard, this multicenter randomized controlled trial (RCT) aims to furtherly evaluate the efficacy and safety of naotaifang capsules on hypertensive cerebral small vessel disease. Methods: This study is a multicenter, randomized, double-blind, placebo-controlled clinical trial. A total of 388 eligible subjects were recruited from the First Hospital of Hunan University of Chinese Medicine, Hunan Academy of Chinese Medicine Affiliated Hospital, the First Hospital of Shaoyang University, the First Traditional Chinese Medicine Hospital of Changde, and Jiangmen Wuyi Hospital of Traditional Chinese Medicine from July 2020 to April 2022. After a 4-week run-in period, all participants were divided into the intervention group (represented by Y-T, N-T) and control group (represented by Y-C, N-C); using a stratified block randomized method based on the presence or absence of brain damage symptoms in hypertensive cerebral small vessel disease (represented by Y and N). The Y-T and N-T groups were administered different doses of naotaifang capsules, whereas Y-C and N-C groups received placebo treatment. These four groups received the treatments for 6 months. The primary outcome included Fazekas scores and dilated Virchow-robin spaces (dVRS) grades on magnetic resonance imaging (MRI). The secondary outcomes included the number of lacunar infarctions (LI) and cerebral microbleeds (CMB) on magnetic resonance imaging, clinical blood pressure (BP) level, traditional Chinese medicine (TCM) syndrome scores, mini-mental state examination (MMSE) scale, and safety outcomes. Fazekas scores, dilated Virchow-robin spaces grades, and the number of lacunar infarctions and cerebral microbleeds on magnetic resonance imaging were tested before enrollment and after 6 months of treatment. The clinical blood pressure level, traditional Chinese medicine syndrome scores, mini-mental state examination scale and safety outcomes were tested before enrollment, after 3-month, 6-month treatment and 12th-month follow-up respectively. Conclusion: The protocol will comfirm whether naotaifang capsules reduce Fazekas scores, dilated Virchow-robin spaces grades, and the number of lacunar infarctions and cerebral microbleeds, clinical blood pressure, increase mini-mental state examination scores, traditional Chinese medicine syndrome scores of Qi deficiency and blood stasis (QDBS), and improve the quality of life of subjects. The consolidated evidence from this study will shed light on the benefits of Chinese herbs for hypertensive cerebral small vessel disease, such as nourishing qi, promoting blood circulation and removing blood stasis, and dredging collaterals. However, additional clinical trials with large samples and long intervention periods will be required for in-depth research. Clinical Trial registration: www.chictr.org.cn, identifier ChiCTR1900024524.

[Preventive and therapeutic effects of Keluoxin Capsules on early diabetic retinopathy in db/db mice].

The aim of this paper was to investigate the preventive effects of Keluoxin Capsules(KLX) on diabetic retinopathy in db/db mice. One hundred male db/db diabetic mice(45-55 g, 8 weeks) were randomly divided into 5 groups(model, KLX low dose, KLX middle dose, KLX high dose, Dobesilate) and 20 male C57 BL/KsJdb~(+/+) were taken as control group. Body weight and fasting blood-glucose were detected every week. Mice were administrated with saline(control and model group), KLX(780, 1 560, 3 120 mg·kg~(-1)·d~(-1), ig), Dobesilate(195 mg·kg~(-1)·d~(-1), ig) for 20 weeks, respectively. At the end of the administration, optical coherence tomography, fundus fluorescein angiography and electroretinogram of the retina were measured. The eyeball was extirpated and retina was isolated to make paraffin section, followed by HE staining and glial fibrillary acidic protein(GFAP) immunohistochemistry. The results indicated that KLX has no obvious effect on body weight and fasting blood level in db/db mice. However, KLX could significantly regulate the thickness of retinal ganglion layer and inner plexiform layer. KLX was able to remarkably reduce the quantity of diabetic microvessel. Meanwhile, KLX could notably improve retinal function. Moreover, KLX could observably modulate the cell arrangement and edema in each layer. There was no markable difference in retina according to the immunochemistry assay. In the present study, KLX exert marked preventive effects on diabetic retinopathy in db/db mice, which provided an experimental evidence for clinical use.

Protective Effects of Rhubarb in Rats with Acute Pancreatitis and the Role of Its Active Compound Rhein on Mitochondria of Exocrine Cells.

Da-Cheng-Qi-Decoction (DCQD) has been used in the treatment of acute pancreatitis (AP) in China for many years. The aim of the current study was to examine the principal ingredient rhubarb of DCQD and its potential link to the pancreatic repair effects in rats with AP. The pancreatitis was induced in SD rats by intraperitoneal injections of cerulein. The results showed that rhubarb significantly increased blood perfusion of pancreatic tissue, reversed mitochondrial damage, and promoted pancreatic acinar and stellate cell proliferation. In addition, the rhein (from rhubarb) had high distribution in pancreas tissue and protected mitochondria in AR42J cells via the activation of PI3K/AKT/mTOR signaling pathway and activity inhibition of AMPK (P < 0.05). The results provide some preclinical evidence on the protective effects of DCQD for the treatment of acute pancreatitis. Rhein is regarded to be the active compound of rhubarb and can be expected to be a new compound for the treatment of AP.

Isolariciresinol-9'-O-α-L-arabinofuranoside protects against hydrogen peroxide­induced apoptosis of human umbilical vein endothelial cells via a PI3K/Akt/Bad­dependent pathway.

Isolariciresinol-9'-O-α-L-arabinofuranoside (MWS­19) isolated from Pinus massoniana Lamb. Fresh pine needles is the major ingredient of the Songling Xuemaikang capsule therapy used for hypertension. The present study aimed to investigate the effects and underlying mechanisms of MWS­19 on hydrogen peroxide (H2O2)­induced apoptosis in human umbilical vein endothelial cells (HUVECs). To investigate the effect of MWS­19 on apoptosis in HUVECs, an oxidative stress­induced apoptosis model was established in HUVECs using H2O2, and the present study performed Hoechst 33258 staining and a Cell Counting Kit­8 (CCK­8) assay. Furthermore, western blot analysis was also performed to investigate the underlying mechanism of the effects of MWS­19 on the model. The results demonstrated that MWS­19 reversed the effects of H2O2 on cell apoptosis at a concentration range of 15.6­250 µg/ml, with dose­dependent increases in cell growth. Hoechst staining indicated that 500 µM H2O2 induced HUVEC apoptosis, and MWS­19 markedly protected HUVECs against apoptosis at 31.3, 62.5 and 125 µg/ml. Furthermore, the protein expression of phosphatidylinositol 3­kinase (PI3K), phosphorylated­Akt and Bcl­2­associated agonist of cell death (Bad) were increased, and reduced caspase­3 activation was observed, following treatment with MWS­19 in H2O2­treated HUVECs. Additionally, the PI3K inhibitor wortmannin attenuated PI3K/Akt/Bad signaling induced by MWS­19 treatment and neutralized the effect of MWS­19 on the growth of HUVECs. In conclusion, the results of the present study indicate that MWS­19 may protect against H2O2­induced HUVEC apoptosis via the PI3K/Akt/Bad signaling pathway. MWS­19 may serve an important role in the prevention of oxidative damage in vascular endothelial cells in hypertension patients.

[Effects of stocking density and exchanging water frequency on growth and inner quality of Whitmania pigra].

The effects of stocking density and exchanging water frequency on growth, digestive enzyme activity, anti-oxidative enzyme and inner quality of Whitmania pigra Whitman were evaluated with corresponding measures. The results showed that the eventual biomass, specific growth rate, gained weight rate, activities of amylase, lipase, protease, SOD, CAT, and ALP correlated positively with stocking density and negatively with exchanging water frequency (P<0.05). Exchanging water frequency had negative correlation with ammonia nitrogen, nitrite, and hydrogen sulfide while revealed positive correlation with dissolved oxygen in the water. Stocking density and exchanging water frequency showed no significant effects on the contents of moisture, total ash, and acid-insoluble ash. It suggested that the optimum stocking density was 7.5 million per hectare and the appropriate exchanging water interval was 72 h.

Compound danshen tablet ameliorated aß25-35-induced spatial memory impairment in mice via rescuing imbalance between cytokines and neurotrophins.

BACKGROUND: Compound Danshen Tablet (CDT), a Traditional Chinese Medicine, has recently been reported to improve spatial cognition in a rat model of Alzheimer's disease. However, in vivo neuroprotective mechanism of the CDT in models of spatial memory impairment is not yet evaluated. The present study is aimed to elucidate the cellular mechanism of CDT on Aß25-35-induced cognitive impairment in mice. METHODS: Mice were randomly divided into 5 groups: the control group (sham operated), the Aß25-35 treated group, the positive drug group, and large and small dosage of the CDT groups, respectively. CDT was administered at a dose of 0.81 g/kg and 0.405 g/kg for 3 weeks. The mice in the positive drug group were treated with 0.4 mg/kg of Huperzine A, whereas the mice of the control and Aß25-35 treated groups were administrated orally with equivalent saline. After 7 days of preventive treatment, mice were subjected to lateral ventricle injection of Aß25-35 to establish the mice model of Alzheimer's disease. Spatial memory impairment was evaluated by Morris water maze test. Choline acetyltransferase (ChAT) contents in hippocampus and cortex were quantified by ELISA. The levels of cytokines, receptor of activated protein kinase C1 (RACK1) and brain-derived neurotrophic factor (BDNF) in hippocampus were measured by RT-PCR and ELISA. RESULTS: The results showed that Aß25-35 caused spatial memory impairment as demonstrated by performance in the Morris water maze test. CDT was able to confer a significant improvement in spatial memory, and protect mice from Aß25-35-induced neurotoxicity. Additionally, CDT also inhibited the increase of TNF-α and IL-6 level, and increased the expression of choline acetyltransferase (ChAT), receptor of activated protein kinase C1 (RACK1) and brain-derived neurotrophic factor (BDNF) in brain as compared to model mice. CONCLUSION: These findings strongly implicate that CDT may be a useful treatment against learning and memory deficits in mice by rescuing imbalance between cytokines and neurotrophins.

Inhibitory effects of ß,ß-dimethylacrylshikonin on hepatocellular carcinoma in vitro and in vivo.

ß,ß-Dimethylacrylshikonin is one of the most abundant naphthoquinones in the root extracts of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), which have been reported to have antitumor effects. This study evaluated the antiproliferative activity of ß,ß-dimethylacrylshikonin on human hepatocellular carcinoma (HCC) cells both in vitro and in vivo. In vitro, the MTT assay showed that ß,ß-dimethylacrylshikonin inhibited the proliferation of SMMC-7721 cells in both dose- and time-dependent manners with its 50% inhibitory concentration (IC(50) ) at 48 h being 15.01 ± 0.76 µg/mL. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and Hoechst staining detected the characteristics of cell apoptosis in ß,ß-dimethylacrylshikonin-treated cells and the apoptotic rates of treated groups were increased in a dose-dependent manner. Flow cytometric analysis revealed that ß,ß-dimethylacrylshikonin could block the cell cycle arrest at G2 phase. Furthermore, ß,ß-dimethylacrylshikonin down-regulated the mRNA and protein expression of Bcl-2 but up-regulated that of Bax. The cleaved caspase-3 protein was also detected in treated cells. The experiment in vivo showed that ß,ß-dimethylacrylshikonin significantly suppressed the growth of H(22) transplantable hepatoma, and induced the activation of caspase-3 determined by immunohistochemistry. The results indicate that ß,ß-dimethylacrylshikonin has significant antitumor effects on hepatocellular carcinoma both in vitro and in vivo.

Calycosin stimulates proliferation of estrogen receptor-positive human breast cancer cells through downregulation of Bax gene expression and upregulation of Bcl-2 gene expression at low concentrations.

BACKGROUND: Calycosin is one of main components in the herb radix astragali and is considered a typical phytoestrogen. It has either estrogenic or antiestrogenic effects that mainly depend on estrogen levels in vivo. This study investigated the effects and mechanisms of calycosin on estrogen receptor (ER)-positive human breast cancer (MCF-7) cells in vitro. METHODS: ER-positive MCF-7 cells were treated with different concentrations of calycosin. Effects of calycosin on the proliferation of ER-positive MCF-7 cells were determined by the MTT assay. Apoptosis in these treated cells was examined by flow cytometry. The mRNA and protein levels of Bcl-2 and Bax in these treated cells were also determined by reverse-transcription polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: Compared with the vehicle control, calycosin stimulated proliferation of ER-positive MCF-7 cells at low concentrations (2, 4, and 8 µmol/L). Furthermore, at these concentrations, calycosin decreased the percentage of early apoptosis in MCF-7 cells, downregulated mRNA and protein levels of Bax, and upregulated those of Bcl-2 at low concentrations. On the other hand, calycosin at higher concentrations (16 and 32 µmol/L) inhibited cell proliferation. CONCLUSION: At relatively low concentrations, calycosin has stimulatory effects on the proliferation of MCF-7 cells, with the estrogenic effect the mechanism.