Aim To investigate the protective effect of Jujing formula on retina of mice with dry age-related macular degeneration(AMD).Methods The mouse model of dry AMD was induced by intraperitoneal in-jection of sodium iodate,and the prognosis was given to the Jujing formula.Retinal thickness was detected by optical coherence tomography(OCT),the retinal morphological changes were observed by hematoxylin-eosin(HE)staining,and the apoptosis of retinal cells was detected by in situ terminal transferase labeling(TUNEL)staining.Combination of tumor necrosis fac-tor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1 β)in eyeballs and serum,superoxide dis-mutase(SOD),glutathione(GSH)and malondialde-hyde(MDA)were evaluated to assess the protective effects of Jujing formula on retinal injury in mice with dry AMD.Results The results of OCT,HE and TUNEL staining showed that Jujing formula significant-ly improved the retinal injury induced by sodium iodate in mice with dry AMD,increased the retinal thickness(P＜0.05),reduced the apoptosis of retinal cells(P＜0.01),and increased the levels of GSH,IL-6 and SOD activity in eyeballs and serum(P＜0.01).The levels of TNF-α,IL-6,IL-1β and MDA were reduced(P＜0.01).Conclusions Jujing formula has certain therapeutic effects on retinal injury in dry AMD,which may be related to inhibiting inflammatory response and enhancing antioxidant capacity.

OBJECTIVE: To evaluate the efficacy and safety of Cidan Capsule combined with adjuvant transarterial chemoembolization (TACE) in patients with a high risk of early recurrence after curative resection of hepatocellular carcinoma (HCC). METHODS: A multicenter, randomized controlled trial was conducted in patients with high-risk recurrence factors after curative resection of HCC from 9 medical centers between July 2014 and July 2018. Totally 249 patients were randomly assigned to TACE with or without Cidan Capsule administration groups by stratified block in a 1:1 ratio. Postoperative adjuvant TACE was given 4-5 weeks after hepatic resection in both groups. Additionally, 125 patients in the TACE plus Cidan group were administrated Cidan Capsule (0.27 g/capsule, 5 capsules every time, 4 times a day) for 6 months with a 24-month follow-up. Primary endpoints included disease-free survival (DFS) and tumor recurrence rate (TRR). Secondary endpoint was overall survival (OS). Any drug-related adverse events (AEs) were observed and recorded. RESULTS: As the data cutoff in July 9th, 2018, the median DFS was not reached in the TACE plus Cidan group and 234.0 days in the TACE group (hazard ratio, 0.420, 95% confidence interval, 0.290-0.608; P<0.01). The 1- and 2-year TRR in the TACE plus Cidan and TACE groups were 31.5%, 37.1%, and 60.8%, 63.4%, respectively (P<0.01). Median OS was not reached in both groups. The 1- and 2-year OS rates in TACE plus Cidan and TACE groups were 98.4%, 98.4%, and 89.5%, 87.9%, respectively (P<0.05). The most common grade 3-4 AEs included fatigue, abdominal pain, lumbar pain, and nausea. One serious AE was reported in 1 patient in the TACE plus Cidan group, the death was due to retroperitoneal mass hemorrhage and hemorrhagic shock, and was not related to study drug. CONCLUSIONS Cidan Capsule in combination with TACE can reduce the incidence of early recurrence in HCC patients at high-risk of recurrence after radical hepatectomy and may be an appropriate option in postoperative anti-recurrence treatment. (Registration No. NCT02253511).

OBJECTIVE: To investigate the factors related to renal impairment in patients with diabetic kidney disease (DKD) from the perspective of integrated Chinese and Western medicine. METHODS: Totally 492 patients with DKD in 8 Chinese hospitals from October 2017 to July 2019 were included. According to Kidney Disease Improving Global Outcomes (KDIGO) staging guidelines, patients were divided into a chronic kidney disease (CKD) 1-3 group and a CKD 4-5 group. Clinical data were collected, and logistic regression was used to analyze the factors related to different CKD stages in DKD patients. RESULTS: Demographically, male was a factor related to increased CKD staging in patients with DKD (OR=3.100, P=0.002). In clinical characteristics, course of diabetes >60 months (OR=3.562, P=0.010), anemia (OR=4.176, P<0.001), hyperuricemia (OR=3.352, P<0.001), massive albuminuria (OR=4.058, P=0.002), atherosclerosis (OR=2.153, P=0.007) and blood deficiency syndrome (OR=1.945, P=0.020) were factors related to increased CKD staging in patients with DKD. CONCLUSIONS Male, course of diabetes >60 months, anemia, hyperuricemia, massive proteinuria, atherosclerosis, and blood deficiency syndrome might indicate more severe degree of renal function damage in patients with DKD. (Registration No. NCT03865914).
Humans ; Male ; Diabetes Mellitus, Type 2 ; Diabetic Nephropathies ; Hyperuricemia ; Kidney ; Proteinuria ; Renal Insufficiency, Chronic/complications*

Aging can cause degenerative changes in the function of multiple tissues and organs in the body. Gastrointestinal diseases and intestinal dysfunction are very common in the elderly people. The purpose of this study is to explore the effect of the total extract of Astragalus membranaceus (Fisch.) Bge. on intestinal function and gut microbiota homeostasis in natural aging mice, which will provide clues for further mechanism study. The natural aging mice model is established and animal experiments follow the regulations of the Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine. The overall health of the mice was evaluated by the "frailty index" scoring method. The intestinal absorption and transport function were measured by detecting intestinal glucose absorption capacity, transport time, lipase and amylase activities of aging mice. Intestinal inflammation was assessed by detecting inflammatory cytokines by enzyme-linked immunosorbent assay (ELISA). The pathological changes in the intestines of aging mice were tested by hematoxylin-eosin (H&E) staining and alizarin blue (AB) staining. The qRT-PCR method was used to explore the gene transcription level related with the proliferation and differentiation of intestinal stem cells. Microbiota analysis based on 16S rDNA were used to evaluate the composition of gut microbiota. The results showed that Astragalus had a tendency to reduce the "frailty index" of aging mice, but did not show a significant difference. In some indicators of aging phenotype, Astragalus has the most significant effect on hair loss and physical fitness. In terms of intestinal function, Astragalus could increase intestinal glucose absorption capacity, shorten intestinal transportation time and promote lipase secretion in aging mice. The levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-‍α) in the aging intestinal tissue were reduced after Astragalus administration. Astragalus also ameliorated the pathological degeneration of the intestinal tissue of aging mice by increasing the length of small intestinal villi, the thickness of colonic mucosa and goblet cell number. In addition, Astragalus elevated the expression of genes associated with the proliferation and differentiation in jejunum and modulated gut microbiota, especially restoring the abundance of Lachnospiraceae. Taken together, the above research results demonstrate the total extract of Astragalus as a key factor improving the intestinal function and gut microbiota homeostasis of aging mice.

In this study, the ameliorative effects of Flos Abelmoschus manihot on mice with chronic inflammatory bowel disease (IBD) were investigated and its effects on the structure of the intestinal flora as well as the lipid profile in feces of IBD mice were analyzed. All animal welfare and experimental procedures followed the regulations of the Animal Ethics Committee of Nanjing University of Chinese medicine. A mouse model with chronic IBD induced by dextran sulfate sodium (DSS) was used to evaluate changes in body weight, disease activity index (DAI), colonic histopathological damage as well as gene expression levels of inflammatory factors in the colon. Fecal samples from mice in each group were collected and subjected to Illumina high-throughput sequencing to detect the abundance of intestinal flora; samples were analyzed by UHPLC-Q-Exactive^® HF Quadrupole-Orbitrap^® of untargeted lipidomics, which detects lipid content in feces. Administration of Flos Abelmoschus manihot could significantly restore the body weight and ameliorate colonic histopathological damage in IBD mice. Sequencing of the gut microbiota revealed that the species diversity and richness of the gut microbiota in IBD mice were decreased, with a significant increase in the abundance of Verrucomicrobia and a significant decrease in the abundance of Bacteroidetes; Flos Abelmoschus manihot significantly increased the richness and diversity of intestinal microbiota in IBD mice, increased the number of taxa species at each level, and restored the abundance of bacteria in the phylum Bacteroidetes. Analysis of fecal lipid profiles identified the most significant changes in sphingolipid and glycerophospholipid metabolic pathways in IBD mice, with Flos Abelmoschus manihot inhibiting ceramide and sphingomyelin synthesis in sphingolipid metabolism. In summary, Flos Abelmoschus manihot can effectively improve the disease condition of mice with chronic IBD, and it has the effect of regulating intestinal flora homeostasis and lipid metabolism, but the related mechanism between the two still needs to be deeply explored.

The purpose of this research is to study the effect of small molecule compound piceatannol (PIC) on host inflammation in adenine induced chronic kidney disease (CKD) mice, and then to explore its mechanism based on the regulation of gut microbiota. All procedures were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine. The level of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected by enzyme linked immunosorbent assay (ELISA); UPLC-TQ/MS technology was used to monitor the level of proinflammatory uremic toxin indoxyl sulfate (IS) and p-cresol sulfate (PCS); the expression of occludin was tested by Western blot; in vitro anaerobic culture of gut bacteria was used to produce indole; the abundance of gut microbiota was evaluated by 16S rDNA sequencing. The results showed that PIC had no effect on inflammatory infiltration in kidney tissue of CKD mice, but could decrease IL-6 level in blood and IL-6/TNF-α level in colon tissue. PIC did not improve intestinal occludin protein expression in CKD mice; while it could significantly reduce the levels of IS and PCS in blood and liver of CKD mice. Further mechanism studies showed that PIC could inhibit the synthesis of IS precursor indole in gut bacteria. Moreover, PIC could decrease the abundance of gut bacteria which producing uremic toxin, such as reducing the abundance of indole and p-cresol producing gut bacteria. In conclusion, PIC could regulate gut microbiota and inhibit the synthesis of uremic toxin precursor, thereafter reducing the accumulation of IS and PCS in vivo, ultimately relieving the inflammation of CKD mice.

BACKGROUND: Shenyankangfu Tablet (SYKFT) is a Chinese patent medicine that has been used widely to decrease proteinuria and the progression of chronic kidney disease. OBJECTIVE: This trial compared the efficacy and safety of SYKFT, for the control of proteinuria in primary glomerulonephritis patients, against the standard drug, losartan potassium. DESIGN, SETTING, PARTICIPANTS AND INTERVENTION: This was a multicenter, double-blind, randomized, controlled clinical trial. Primary glomerulonephritis patients, aged 18-70 years, with blood pressure ≤ 140/90 mmHg, estimated glomerular filtration rate (eGFR) ≥ 45 mL/min per 1.73 m MAIN OUTCOME MEASURES: The primary outcome was change in the 24-hour proteinuria level, after 48 weeks of treatment. RESULTS: A total of 735 participants were enrolled. The percent decline of urine protein quantification in the SYKFT group after 48 weeks was 8.78% ± 2.56% (P = 0.006) more than that in the losartan 50 mg group, which was 0.51% ± 2.54% (P = 1.000) less than that in the losartan 100 mg group. Compared with the losartan potassium 50 mg group, the SYKFT plus losartan potassium 50 mg group had a 13.39% ± 2.49% (P < 0.001) greater reduction in urine protein level. Compared with the losartan potassium 100 mg group, the SYKFT plus losartan potassium 100 mg group had a 9.77% ± 2.52% (P = 0.001) greater reduction in urine protein. With a superiority threshold of 15%, neither was statistically significant. eGFR, serum creatinine and serum albumin from the baseline did not change statistically significant. The average change in TCM syndrome score between the patients who took SYKFT (-3.00 [-6.00, -2.00]) and who did not take SYKFT (-2.00 [-5.00, 0]) was statistically significant (P = 0.003). No obvious adverse reactions were observed in any group. CONCLUSION: SYKFT decreased the proteinuria and improved the TCM syndrome scores of primary glomerulonephritis patients, with no change in the rate of decrease in the eGFR. SYKFT plus losartan potassium therapy decreased proteinuria more than losartan potassium therapy alone. TRIAL REGISTRATION NUMBER NCT02063100 on ClinicalTrials.gov.

Objective To establish a new patient-derived xenograft (PDX) model of human liver cancer by inoculating the complex of human primary liver cancer cells and a novel microcarrier (microcarrier 6) into mice with normal immune function. Methods Primary liver cancer cells were isolated and extracted from the fresh human liver cancer tissue of five patients and were then co-cultured with microcarrier 6 to construct a three-dimensional tumor cell culture model in vitro . According to the type of graft, 75 male C57BL/6 mice were divided into cell control group, microcarrier control group, and experimental group (each sample corresponded to three groups, with 15 groups in total and 5 mice in each group). The liver cancer cell-microcarrier complex was implanted into the mice by subcutaneous inoculation, and tumor formation time, tumor formation rate, and histopathological manifestations were observed. The Fisher's exact test was used for comparison of categorical data between two groups. Results As for the liver cancer cells from the five patients, tumor formation was observed in the mice corresponding to three patients. In these three experiments, tumor formation was not observed in the control groups and was only observed in the experimental groups, and 12 of the 15 mice in the experimental groups had successful tumor formation, with a tumor formation rate as high as 80%, which was significantly different from that in the cell control groups and the microcarrier control groups (all P < 0.05). The tumor formation time was 5-7 days; the xenograft tumor grew rapidly, and HE staining showed nested or flaky cells with obvious heteromorphism, with the presence of pathological mitosis; immunohistochemical staining showed positive CK8/18, Hep, and Gpc-3, which was in accordance with the characteristics of human liver cancer cells. Conclusion This experiment successfully establishes a new PDX model of human liver cancer based on the complex of microcarrier 6 and human primary liver cancer cells in mice with normal immunity. This model can be used to better elucidate the mechanism of the development and progression of liver cancer in the body with normal immunity, and besides, it also provides a new animal model with higher value for the precise treatment of liver cancer.

Objective To investigate the real correlation between embryo density and developmental outcome of cleavage embryos on day 3 by adjusting covariates. Methods Data of 1196 embryos from 206 couples undergoing in vitro fertilization and embryo transfer(IVF-ET) treatment were retrospectively analyzed. Embryos were hypoxia-cultivated in a fixed 30 μl microdrop in culture dishes. Embryo quality on day 3 was evaluated and proportion of good quality cleavage embryos on day 3 was used as the endpoint. Maternal age, paternal age, antral follicles, level of anti-mullerian hormone (AMH), type of infertility, controlled ovarian stimulation(COS) protocol, length of stimulation, number of retrieved oocytes, and type of insemination were chosen as covariates. Results A total of 1196 embryos were included and analyzed. Three embryo densities were routinely used: 30 μl/ embryo [1 embryo/ (30 μl·drop)], 15 μl/ embryo [2 embryos / (30 μl·drop)] and 10 μl/ embryo [3 embryos/ (30 μl·drop)]. The number of embryos assigned into these three groups were 434, 646 and 116 embryos separately. The average proportion of good quality embryos on day 3 in 10 μl/ embryo group were lower than that in both 15 and 30 μl/ embryo group (29. 31% vs 40. 25%,P<0. 05;29. 31% vs 42. 17%,P<0. 05), and it was comparable between groups of 15 and 30 μl/ embryo (40. 25% vs 42. 17%, P > 0. 05). In the regression analysis, without adjusting any covariables, the good quality embryos rate on day 3 of the 10 μl/ embryo group was 43% lower than that of the 30 μl/ embryo group (0. 57, 95% CI 0. 32, 1. 03), and there was no statistical difference (P>0. 05). In the minimum-adjusted model 2 (adjusted the level of AMH and type of insemination), proportion of good quality embryos on day 3 in group of 10 μl/ embryo significantly decreased by 51% (0. 49, 95% CI 0. 27,0. 90, P < 0. 05) compared with that in group of 30 μl/ embryo. Conclusion In a 30 μl microdrop, compared with individual cul turing, group culturing with the density of 10 μl / embryo did not benefit the development of the cleavage embryos and 30 μl / embryo was the optimal embryo density.

Uremic toxins are harmful substances that accumulate in the body when the renal function declines in patients with chronic kidney disease (CKD). It is an important factor contributing to accelerated progression of CKD. There is no effective treatment for reducing uremic toxins. As an extensively used medicine for treatment of CKD in the clinic, Huangkui capsule is effective but the mechanism of its action remains unclear. This study investigated the effect of Huangkui on the accumulation of uremic toxins in CKD rats, with the discussion about its mechanism of action. UPLC-TQ/MS was used to detect the accumulation of uremic toxins in CKD rats after oral gavage with Huangkui. 16S rDNA sequencing technology was used to analyze the gut bacteria composition in rats. HPLC-FLD was used to detect the uremic toxins and their molecular precursors in feces. The effect and mechanism of Huangkui on the uremic toxin precursor in gut bacteria were studied by anaerobic culture system in vitro. All procedures were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine. The results showed that Huangkui (0.675 g·kg^-1) could effectively inhibit the accumulation of uremic toxin indoxyl sulfate (IS) in CKD rats, with IS concentration in rat's plasma, liver and kidney decreased by 49.5%, 68.9% and 40.6%, respectively. Huangkui didn't affect the metabolic pathway of IS in host liver, didn't intervene the process of the IS precursor molecule indole conversion to IS. Instead, Huangkui significantly decreased the indole content in gut, with the indole in CKD rat's feces decreased by 46.4%, suggesting that the gut bacteria may be a target for intervene IS biosynthesis by Huangkui. Huangkui didn't affect the abundance of enterobacteriaceae bacteria (the main gut flora of indole synthesis) in CKD rats, suggesting that Huangkui didn't interfere with indole biosynthesis by directly affecting the abundance of indole synthesis related bacteria. Huangkui at 4 000, 400, 40, and 4 μg·mL^-1 showed a dose-dependent inhibition of the indole production by gut bacteria in vitro. The bacteria tryptophan transport concentration decreased from 83.4 μmol·L^-1 to 43.6 μmol·L^-1 after co-incubated with Huangkui for 12 h, suggesting that Huangkui inhibited indole production of gut bacteria by interfering with tryptophan transportation. These results indicate that gut bacteria may be a potential target for alleviation of uremic toxin accumulation and for delaying CKD progression.