Objective To observe the effects of melatonin on autophagy in cortical neurons of neonatal rats with hypoxic-ischemic brain damage(HIBD)and to explore its mechanisms via the PI3K/AKT signaling pathway,aiming to provide a basis for the clinical application of melatonin.Methods Seven-day-old Sprague-Dawley neonatal rats were randomly divided into a sham operation group,an HIBD group,and a melatonin group(n=9 each).The neonatal rat HIBD model was established using the classic Rice-Vannucci method.Neuronal morphology in the neonatal rat cerebral cortex was observed with hematoxylin-eosin staining and Nissl staining.Autophagy-related protein levels of microtubule-associated protein 1 light chain 3(LC3)and Beclin-1 were detected by immunofluorescence staining and Western blot analysis.Phosphorylated phosphoinositide 3-kinase(p-PI3K)and phosphorylated protein kinase B(p-AKT)protein expression levels were measured by immunohistochemistry and Western blot.The correlation between autophagy and the PI3K pathway in the melatonin group and the HIBD group was analyzed using Pearson correlation analysis.Results Twenty-four hours post-modeling,neurons in the sham operation group displayed normal size and orderly arrangement.In contrast,neurons in the HIBD group showed swelling and disorderly arrangement,while those in the melatonin group had relatively normal morphology and more orderly arrangement.Nissl bodies were normal in the sham operation group but distorted in the HIBD group;however,they remained relatively intact in the melatonin group.The average fluorescence intensity of LC3 and Beclin-1 was higher in the HIBD group compared to the sham operation group,but was reduced in the melatonin group compared to the HIBD group(P<0.05).The number of p-PI3K+and p-AKT+cells decreased in the HIBD group compared to the sham operation group but increased in the melatonin group compared to the HIBD group(P<0.05).LC3 and Beclin-1 protein expression levels were higher,and p-PI3K and p-AKT levels were lower in the HIBD group compared to the sham operation group(P<0.05);however,in the melatonin group,LC3 and Beclin-1 levels decreased,and p-PI3K and p-AKT increased compared to the HIBD group(P<0.05).The correlation analysis results showed that the difference of the mean fluorescence intensity of LC3 and Beclin-1 protein in the injured cerebral cortex between the melatonin and HIBD groups was negatively correlated with the difference of the number of p-PI3K+and p-AKT+cells between the two groups(P<0.05).Conclusions Melatonin can inhibit excessive autophagy in cortical neurons of neonatal rats with HIBD,thereby alleviating HIBD.This mechanism is associated with the PI3K/AKT pathway.

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*

Colorectal cancer (CRC) is a common malignancy burdening people globally, with increasing morbidity and mortality nowadays, due to the alternation in the diet type and lifestyle in modern society. Berberine, a type of benzylisoquinoline alkaloid, is widely present in numerous medicinal plants, particularly including Coptidis Rhizoma. Mounting evidence reveals that berberine possesses an array of pharmacological effects, such as anti-inflammation, anti-bacterium, anti-cancer, anti-diabetes mellitus and so on. In particular, berberine exhibits substantial inhibition on various types of cancers including CRC. Hereby, we sought to systematically review the suppressive effect of berberine on CRC through the diminishment of the proliferation and metastasis, induction of apoptosis, arrest of cell cycle, regulation of inflammatory reaction, the reverse of chemotherapeutic resistance and restoration of gut microbiota in CRC, so as to shed light on the in-depth mechanisms underlying the treatment of CRC with berberine in the clinical setting.

ObjectiveTo explore the underlying mechanism of bile acids and metabolites as well as the key metabolic pathways and important endogenous targets in prehypertension. MethodThe metabolic mechanism of prehypertension was explored with non-targeted metabolomics combined with network analysis. The serum metabolomics of patients with prehypertension was analyzed by ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. The relevant biological functions and signal targets were predicted and generated by network analysis. Finally，the predicted targets of this important pathway were verified by in vitro experiments，and the relevant information was verified by enzyme-linked immunosorbent assay （ELISA） and Western blot. ResultAs revealed by non-targeted metabolomics，there were 64 potential biomarkers and 13 metabolic pathways in the normal group，the prehypertension group， and the hypertension group. The results of network analysis and biological verification showed that the occurrence of prehypertension was related to vascular inflammation caused by the abnormal metabolism of bile acids and aromatic amino acids. Bile acid metabolism plays an important role in the occurrence and development of prehypertension by regulating the vascular inflammatory response. Amino acid N-acyltransferase，myeloperoxidase， and bile acid downstream receptor TGR5 are critical in the changes of the metabolic network. ConclusionIn prehypertension，bile acids are presumedly involved in regulating vascular inflammation， resulting in damage to blood vessels in prehypertension.

Traditional Chinese medicine polysaccharides is a biologically active ingredient that is not easy to be digested. It is fermented by intestinal microflora to promote qualitative and selective changes in the composition of the intestinal microbiome, which often result in beneficial effects on the health of the host. People call it "prebiotics". In this review, we systematically summarized the anti-diabetic effect of traditional Chinese medicine polysaccharides. These polysaccharides regulate the metabolism of sugar and lipids by inter-influence with the intestinal microflora, and maintain human health, while improving type 2 diabetes-like symptoms such as high blood glucose, and abnormal glucose and lipid metabolism.
Blood Glucose/metabolism* ; Diabetes Mellitus, Type 2/metabolism* ; Humans ; Lipids ; Medicine, Chinese Traditional ; Polysaccharides/pharmacology* ; Probiotics/therapeutic use*

OBJECTIVE: To obtain the subtypes of the clinical hypertension population based on symptoms and to explore the relationship between hypertension and comorbidities. METHODS: The data set was collected from the Chinese medicine (CM) electronic medical records of 33,458 hypertension inpatients in the Affiliated Hospital of Shandong University of Traditional Chinese Medicine between July 2014 and May 2017. Then, a hypertension disease comorbidity network (HDCN) was built to investigate the complicated associations between hypertension and their comorbidities. Moreover, a hypertension patient similarity network (HPSN) was constructed with patients' shared symptoms, and 7 main hypertension patient subgroups were identified from HPSN with a community detection method to exhibit the characteristics of clinical phenotypes and molecular mechanisms. In addition, the significant symptoms, diseases, CM syndromes and pathways of each main patient subgroup were obtained by enrichment analysis. RESULTS: The significant symptoms and diseases of these patient subgroups were associated with different damaged target organs of hypertension. Additionally, the specific phenotypic features (symptoms, diseases, and CM syndromes) were consistent with specific molecular features (pathways) in the same patient subgroup. CONCLUSION The utility and comprehensiveness of disease classification based on community detection of patient networks using shared CM symptom phenotypes showed the importance of hypertension patient subgroups.

To prove that ursolic acid(UA)could activate the autophagy of colorectal cancer HCT116 cells by inhibiting hedgehog signaling pathway. The effect of UA on the viability of HCT116 cells was determined by MTT assay. The effect of UA on the proliferation and migration of HCT116 cells was detected by crystal violet staining and scratch test. In the study on autophagy, the time points were screened out first: the autophagy fluorescence intensity of UA acting on HCT116 at different time points were detected by Cell Meter~(TM) Autophagy Assay Kit; Western blot was used to detect the expression of autophagy protein P62 at different time points. Then, Cell Meter~(TM) Autophagy Assay Kit was used to detect the effect of UA on autophagy fluorescence intensity of HCT116 cells. The effect of different doses of UA on the expressions of LC3Ⅱ and P62 proteins in HCT116 cells were detected by Western blot. Further, AdPlus-mCherry-GFP-LC3 B adenovirus transfection was used to detect the effects of UA on autophagy flux of HCT116 cells; UA combined with autophagy inhibitor chloroquine(CQ) was used to detect the expression of LC3Ⅱ by Western blot. In terms of mechanism, the effect of UA on hedgehog signaling pathway-related proteins in HCT116 cells was detected by Western blot. The results showed that UA inhibited the activity, proliferation and migration of HCT116 cells. UA enhanced the fluorescence intensity of autophagy in HCT116 cells, while promoting the expression of LC3Ⅱ and inhibiting the expression of P62, in a time and dose dependent manner. UA activated the autophagy in HCT116 cells, which manifested that UA resulted in the accumulation of fluorescence spots and strengthened the fluorescence intensity of autophagosomes; compared with UA alone, UA combined with autophagy inhibitor CQ promoted the expression of LC3Ⅱ. UA reduced the expressions of PTCH1, GLI1, SMO, SHH and c-Myc in hedgehog signaling pathway, while increased the expression of Sufu. In conclusion, our study showed that UA activated autophagy in colorectal cancer HCT116 cells, which was related to the mechanism in inhibiting hedgehog signaling pathway activity.
Apoptosis ; Autophagy ; Cell Line, Tumor ; Colorectal Neoplasms ; Hedgehog Proteins/genetics* ; Humans ; Signal Transduction ; Triterpenes

OBJECTIVE To investigate the therapeutic effect of scutellarin on colitis-associated cancer (CAC) and its underlying mechanism based on Wnt/β-catenin signaling pathway. METHODS The mouse model of CAC was estab?lished by azomethane oxide (AOM) and sodium dextran sulfate (DSS), followed by scutellarin treatment, with recording the body weight, diarrhea and hematochezia. After sacrificing the mice, the colorectal length and colorectal tumor were assessed. The levels of pro-inflammatory factors TNF-α and IL-6 in mice's sera were measured by the enzyme-linked immunosorbent assay (ELISA). The colorectal lesions were appraised by hematoxylin and eosin (H&E) staining. Theβ-catenin level in CAC tissues was probed by immunofluorescent analysis. The apoptosis-related genes Bax and Bcl-2, and Wnt signaling pathway-related genes β-catenin, GSK-3β, TCF4, c-Myc and cyclin D1 were detected by real-time quantitative RT-PCR (RT-qPCR). Finally, Western blotting analysis (WB) was employed to examine the expressions of the apoptosis and Wnt signaling pathway-related proteins. RESULTS Scutellarin significantly improved AOM/DSS-caused weight loss, colorectal length shortening, and tumor growth in mice (P<0.01). Meanwhile, colorectal lesions could be substantially alleviated by scutellarin. ELISA results showed that the levels of pro-inflammatory factors TNF-αand IL-6 were drastically lessened (P<0.01). Scutellarin also sharply inhibited the nuclear translocation of β-catenin, as evidenced by the reduction in the nuclear level ofβ-catenin protein. In addition, scutellarin attenuated the mRNA expres?sion of Wnt signaling pathway-relatedβ-catenin, TCF4, c-Myc and cyclin D1, whereas it heightened GSK-3βmRNA level. These results were consolidated by WB analysis, which indicated that scutellarin could mitigate the protein levels of phospho-GSK-3β,β-catenin, TCF4, c-Myc and cyclin D1, with the increase in GSK-3β protein in CAC tissue. Moreover, scutellarin could induce the apoptosis of CAC, demonstrated by enhanced expression of Bax and diminished expression of Bcl-2 in both mRNA and protein levels. CONCLUSION Scutellarin may ameliorate colitis-associated colorectal cancer by weakening Wnt/β-catenin signaling cascade.

OBJECTIVE To identify the inhibitory effect of ursolic acid on the colorectal cancer HCT116 cells in vitro and in vivo, and to explore the underlying mechanism. METHODS The smoothened (SMO) gene-silenced human colorectal cancer HCT116hSMO- cell line was established by transfection with the lentivirus carrying SMO shRNA. The cytotoxic effect of ursolic acid on HCT116hSMO-cells was determined by MTT assay. The effect of ursolic acid on the migration of HCT116hSMO- cells was studied by wound healing assay. The effect of ursolic acid on apoptosis of HCT116hSMO-cells was explored by Hoechst33342/PI double staining and flow cytometry. The effects of ursolic acid on the expressions of apoptotic marker gene Bcl-2, Bax, caspase-3 and caspase-9 were measured by real-time quantitative RT-PCR (RT-qPCR) and Western blotting (WB) analysis. RT-qPCR and WB were used to examine the relationship between GLI1, c-Myc expression and PI3K/Akt pathway to further investigate the mechanism of GLI1 activation in HCT116hSMO- cells. The effects of ursolic acid on the expressions of GLI1, p-Akt, Akt, c-Myc, SHH and SUFU of nonca?nonical Hedgehog pathway were evaluated by RT-qPCR and WB assays. Xenograft nude mouse model bearing HCT116hSMO- cells was established and intraperitoneally treated with ursolic acid to investigate the effect on tumor growth in vivo. The body weight and tumor size of mice were assessed regularly every 2 d. The effect of ursolic acid on the apoptosis of tumor tissue was determined by TUNEL assay. The expressions of Bcl-2, Bax, GLI1, p-Akt, Akt, c-Myc, SHH, SUFU mRNA and proteins were measured by RT-qPCR and WB. The levels of Bcl-2, Bax, GLI1, p-Akt, c-Myc and SHH proteins in tumor tissues were also evaluated by immunohistochemistry. RESULTS Ursolic acid significantly inhibited the growth and migration of HCT116hSMO-cells in vitro, compared with the control (P<0.05). Meanwhile, ursolic acid also induced apoptosis of HCT116hSMO- cells in vitro (P<0.05). Furthermore, SC79 (Akt activator) enhanced the expressions of p-Akt, GLI1 and c-Myc, which could be abolished by ursolic acid, and the effect was equal to Akt inhibitor LY294002. The expressions of Bcl-2, GLI1, p-Akt, c-Myc, SHH mRNA and proteins were reduced by ursolic acid, while the levels of Bax and SUFU were increased. Ursolic acid could inhibit the growth and induce the apoptosis of colorectal cancer xeno?graft in vivo. Similarly, lower levels of Bcl-2, GLI1, p-Akt, c-Myc and SHH, and higher expression of Bax and SUFU were noted in ursolic acid-treated mice. CONCLUSION Ursolic acid can inhibit the growth and induce apoptosis of HCT116hSMO- cells both in vitro and in vivo. And the mechanism is related to the suppression of PI3K/Akt-mediated noncanonical Hedgehog signaling pathway.

Pulsatilla chinensis is a widely used traditional Chinese herb, which contains 56 types of chemical constit?uents, mainly including triterpenoid saponins, organic acids, coumarins and lignans. The largest portion of the ingredi?ents in Pulsatilla chinensis is the family of triterpenoid saponins, in which anemoside B4 is the major effective compound and indexing component. The main components of Pulsatilla chinensis can metabolize into a vast array of active prod?ucts in vivo, which play vital roles in its biological activity. Mounting evidence reveals that Pulsatilla chinensis exerts a wide range of therapeutic activities, such as anti-cancer, immunoregulation, anti-inflammation and anti-schistosome, with fewer adverse reactions, via various signaling pathways and multiple targets. It was documented that the active ingre?dient of Pulsatilla chinensis can lessen the drug resistance and synergize the effects of other natural products includ?ing paclitaxel, as well as ameliorate the clinical efficacy of chemical drugs, such as adriamycin. However, Pulsatilla chi?nensis was also reported to be possibly the main cause of hemolysis and chronic liver injury. The efforts should be made to deeply investigate the pharmacological actions and underlying mechanisms of Pulsatilla chinensis, with a focus on the anti-cancer efficacy, and develop new drugs based on the components of Pulsatilla chinensis for future utilization in the clinical setting.