Objective: To investigate whether rosuvastatin acts on lymphatic system and influences lymphatic system-mediated reverse cholesterol transport to play an anti-atherosclerosis role. Methods: Forty-eight apolipoprotein E^-/- mice fed a high fat diet were used to construct the atherosclerosis model. They were randomly divided into 4 groups with 12 rats in each group. They were treated with rosuvastatin, vascular endothelial growth factor-C (VEGF-C) and rosuvastatin+VEGF-C inhibitors as experimental group, and no intervention measures were given in control group. After 8 weeks, aortic plaque area, high density lipoprotein cholesterol (HDL-C) content in lymph fluid, the function of popliteal lymphatic drainage of peripheral Evans blue, and the ability of lymphatic system to transport peripheral cell membrane red fluorescent probes to label high-density lipoprotein (HDL) were detected. Subsequently, the effects of rosuvastatin on proliferation, migration and tubular function of lymphoendothelial cells and the expression of scavenger receptor class B type 1 (SR-B1) on lymphoendothelial cells at different concentrations were detected. Results: Compared with the control group, Rosuvastatin and VEGF-C could reduce the area of aortic atherosclerotic plaque (P<0.05). In addition to rosuvastatin plus VEGF-C inhibitor, the intra-aortic plaque area increased (P<0.05). Compared with the control group, Rosuvastatin could increase the content of HDL-C in lymphatic fluid (P<0.05), enhance the drainage function of lymphatic vessels, and enhance the capacity of HDL in the transport tissue fluid of lymphatic system. Compared with the control group, VEGF-C increased the content of HDL-C in mouse lymph fluid (P<0.01), enhanced the drainage function of popliteal lymphatic canal, and enhanced the ability of lymphatic system to transport HDL. With the addition of VEGF-C inhibitor on the basis of rosuvastatin, the content of HDL-C in lymph fluid was reduced, the drainage of popliteal lymphatic canal was interrupted, and the ability of lymphatic system to transport HDL was reduced. Western blotting showed that rosuvastatin increased the protein expression of SR-B1. Conclusion: Rosuvastatin can promote the proliferation, migration and tube formation of lymphatic endothelial cells. At the same time, SR-B1 expression on lymphatic endothelial cells is promoted, thus enhancing the lymphatic system mediated cholesterol reversal transport and playing the role of anti-atherosclerosis.
Rats ; Mice ; Animals ; Rosuvastatin Calcium/therapeutic use* ; Vascular Endothelial Growth Factor C ; Endothelial Cells/metabolism* ; Atherosclerosis/drug therapy* ; Plaque, Atherosclerotic ; Cholesterol, HDL ; Lymphatic System/metabolism*

BACKGROUND: Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. METHODS: Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. RESULTS: Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. CONCLUSIONS Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.
Animals ; Humans ; Mice ; Apoptosis ; Bone Marrow Cells ; Cell Communication ; Connexin 43/genetics* ; Gap Junctions/metabolism* ; Imatinib Mesylate/therapeutic use* ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology* ; Mesenchymal Stem Cells/metabolism* ; Tumor Microenvironment ; Calcium/metabolism*

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1β, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1β, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1β, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.
Female ; Animals ; Humans ; Mice ; Candidiasis, Vulvovaginal/drug therapy* ; Inflammasomes/genetics* ; Interleukin-18 ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; 1-Butanol/pharmacology* ; Fluconazole/therapeutic use* ; Interleukin 1 Receptor Antagonist Protein/therapeutic use* ; Mice, Inbred C57BL ; Candida albicans ; Cytokines ; Drugs, Chinese Herbal/pharmacology* ; Ethanol ; RNA, Messenger ; Calcium-Binding Proteins/therapeutic use*

OBJECTIVE: To compare the clinical effects of pulpotomy with two kinds of calcium silicate materials, and to evaluate the formation of dentin bridge and pulp calcification after pulpotomy of adult permanent teeth. METHODS: Patients who visited the General Department of Peking University School and Hospital of Stomatology from November 2017 to September 2019 and planned for pulpotomy on permanent premolars and molars with carious exposed pulp were selected. They were randomly divided into two groups. Bioceramic putty material iRoot BP (iRoot group, n=22) and mineral trioxide aggregate MTA (MTA group, n=21) were used as pulp capping agents, respectively. The patients were recalled after one year and two years. The clinical efficacy, dentin bridge index (DBI) and pulp calcification index (PCI) were recorded. Blinding method was used for the patients and evaluators. RESULTS: There was no significant difference in gender, mean age, dentition and tooth position between the two groups (P>0.05). Seven cases were lost during the first year (4 cases in iRoot group and 3 cases in MTA group). In the iRoot group, 1 case had transient sensitivity at the time of 1-year follow-up. The cure rate of the two groups was 100% at the time of 2-year follow-up. The proportion of dentin bridge formation was 38.9% one year after operation, 55.6% two years after operation. The proportion of partial or even complete disappearance of root canal image was 5.6% before operation, 38.9% and 55.6% one and two years after operation, respectively. The difference was statistically significant by rank sum test (P < 0.05). There was no significant difference in dentin bridge formation and pulp calcification between the two groups (P < 0.05). DBI and PCI after operation was as the same as those before operation (44.4% cases of DBI and 25% cases of PCI) or gradually increased (55.6% cases of DBI and 75% cases of PCI). Spearman's nonparametric correlation analysis showed that age was positively correlated with preoperative pulp calcification index (PCI0, P < 0.05), but not with the dentin bridge index (DBI1, DBI2), pulp calcification index (PCI1, PCI2) and the degree of change (DBI2 vs. DBI1, PCI1 vs. PCI0, PCI2 vs. PCI0) 1-year and 2-year after operation (P>0.05). CONCLUSION According to this study, good clinical effects were obtained within 2-year after pulpotomy of adult permanent teeth with MTA and iRoot. In some cases, the root canal system had a tendency of calcification aggravation, and there was no statistical difference in the development of this trend between the two groups.
Humans ; Adult ; Pulpotomy/methods* ; X-Rays ; Calcium Compounds/therapeutic use* ; Dentition, Permanent ; Molar/surgery* ; Treatment Outcome ; Silicates/therapeutic use* ; Aluminum Compounds/therapeutic use* ; Oxides ; Drug Combinations ; Dental Pulp Capping

OBJECTIVE: To investigate the clinical efficacy of acrylic cement (PMMA) mixed with calcium sulfate combined with percutaneous kyphoplasty (PKP) in the treatment of osteoporotic fracture (OVCF). METHODS: The clinical data of 191 patients with OVCF treated with PKP from January 2020 to March 2021 were retrospectively analyzed. Among them, 82 patients with 94 vertebral bodies were treated with PMMA mixed with calcium sulfate as the observation group, and 109 patients with 125 vertebral bodies were treated with pure PMMA as the control group. Among the 82 patients in the observation group, there were 16 males and 66 females, with a mean age of (75.35±11.22) years old, including 36 thoracic vertebrae and 58 lumbar vertebrae. In the control group, there were 109 patients, 22 males and 87 females, with an average age of (74.51±9.21) years old, including 63 thoracic vertebrae and 62 lumbar vertebrae. The visual analog scale (VAS) before operation and 1 day, 3 months and 1 year after operation were calculated. The Oswestry disability index (ODI), Cobb's angle, vertebral body height and the probability of postoperative bone cement leakage were used to analyze the efficacy of the two groups. RESULTS: All the patients were followed up for more than one year. Compared with the control group, there was no significant difference in operation time, bleeding volume and bone cement injection volume between the two groups(P>0.05), while the leakage rate of bone cement was significantly lower in the observation group (P<0.05). In addition, there was no significant difference in VAS, ODI, Cobb angle, and vertebral body height between the two groups before operation, and 1 day, 3 months, and 1 year after operation (P>0.05), but each index was improved compared with that before operation (P<0.05). CONCLUSION PMMA mixed with calcium sulfate has equivalent efficacy in treating OVCF than PMMA alone, but can effectively reduce the probability of cement leakage.
Female ; Male ; Humans ; Aged ; Aged, 80 and over ; Middle Aged ; Polymethyl Methacrylate ; Calcium Sulfate/therapeutic use* ; Osteoporotic Fractures/surgery* ; Bone Cements/therapeutic use* ; Kyphoplasty ; Retrospective Studies ; Lumbar Vertebrae/surgery*

Vitamin D can not only regulate calcium and phosphorus metabolism, but also exert an immunoregulatory effect. Vitamin D deficiency is common in patients with Crohn's disease (CD). Studies have shown that vitamin D is associated with CD and other autoimmune diseases and can improve the condition of patients with CD and promote their recovery by regulating intestinal immunity, repairing the intestinal mucosal barrier, inhibiting intestinal fibrosis, enhancing the response to infliximab, and regulating intestinal microbiota. Exogenous vitamin D supplementation can induce disease remission while increasing the serum level of vitamin D. However, only a few randomized, double-blind, and placebo-controlled trials have investigated the therapeutic effect of vitamin D in CD, and the optimal form of vitamin D supplementation, the specific dosage of vitamin D supplementation, and the optimal serum maintenance concentration of vitamin D remain to be clarified. This article mainly discusses the mechanism of action of vitamin D in CD and the beneficial effect of exogenous vitamin D supplementation on CD.
Humans ; Calcium, Dietary ; Crohn Disease/drug therapy* ; Dietary Supplements ; Infliximab ; Vitamin D/therapeutic use*

OBJECTIVE: To evaluate the clinical characteristics and effectiveness of pulpotomy in mature permanent teeth with bioceramic putty repairmen iRoot and mineral trioxide aggregate (MTA). METHODS: Pulpotomy was performed on mature permanent premolars and molars with carious exposures at the Department of General Dentistry of Peking University School and Hospital of Stomatology, from November 2017 to September 2019. The patients were randomly divided into 2 groups, Group iRoot (n=22) and Group MTA (n=21). In Group iRoot, bioceramic putty repairmen iRoot was used as pulp capping agent, while in Group MTA, mineral trioxide aggregate was used as pulp capping agent. All the patients had signed informed consent forms. The clinical efficacy was evaluated by clinical examinations (temperature and electrical activity test) and imaging examinations 3, 6, and 12 months after surgery. Blinding was used for the patients and evaluators, but due to the obvious differences in the properties of the two pulp capping agents, the blinding method was not used for the treatment provider (the attending physician). RESULTS: There was no significant difference in gender, average age, dentition and tooth position distribution between the two groups (P>0.05). In the study, 7 cases were lost to follow-up 12 months after operation (4 cases in Group iRoot, and 3 cases in Group MTA). One case in each of the two groups had transient sensitivity at the end of the 3-month follow-up, and the pulp vitality was normal at the end of the 6-month follow-up. One case in Group iRoot showed sensitivity at the end of the 12-month follow-up. The success rates of the two groups at the end of 12-month follow-up were 100%, and the cure rates were 94.4% (Group iRoot) and 100% (Group MTA), respectively, and the difference was not statistically significant (P>0.05). No cases in Group iRoot had obvious crown discoloration, while 3 cases in Group MTA had. CONCLUSION The clinical characteristics and effectiveness of pulpotomy in mature permanent teeth with bioceramic putty repairmen iRoot were similar with MTA. Bioceramic putty repairmen iRoot is an acceptable material when used in pulpotomy of mature permanent teeth. Because it is not easy to cause tooth discoloration after treatment and is convenient to operate, bioceramic putty repairmen iRoot has a better clinical application prospect.
Aluminum Compounds/therapeutic use* ; Calcium Compounds/therapeutic use* ; Drug Combinations ; Humans ; Oxides/therapeutic use* ; Pulpotomy ; Silicates/therapeutic use* ; Treatment Outcome

Pseudo-allergic reactions (PARs) widely occur upon application of drugs or functional foods. Anti-pseudo-allergic ingredients from natural products have attracted much attention. This study aimed to investigate anti-pseudo-allergic compounds in licorice. The anti-pseudo-allergic effect of licorice extract was evaluated in rat basophilic leukemia 2H3 (RBL-2H3) cells. Anti-pseudo-allergic compounds were screened by using RBL-2H3 cell extraction and the effects of target components were verified further in RBL-2H3 cells, mouse peritoneal mast cells (MPMCs) and mice. Molecular docking and human MRGPRX2-expressing HEK293T cells (MRGPRX2-HEK293T cells) extraction were performed to determine the potential ligands of MAS-related G protein-coupled receptor-X2 (MRGPRX2), a pivotal target for PARs. Glycyrrhizic acid (GA) and licorice chalcone A (LA) were screened and shown to inhibit Compound48/80-induced degranulation and calcium influx in RBL-2H3 cells. GA and LA also inhibited degranulation in MPMCs and increase of histamine and TNF-α in mice. LA could bind to MRGPRX2, as determined by molecular docking and MRGPRX2-HEK293T cell extraction. Our study provides a strong rationale for using GA and LA as novel treatment options for PARs. LA is a potential ligand of MRGPRX2.
Animals ; Anti-Allergic Agents/therapeutic use* ; Calcium/metabolism* ; Cell Degranulation ; Glycyrrhiza ; HEK293 Cells ; Humans ; Hypersensitivity/drug therapy* ; Mast Cells/metabolism* ; Mice ; Mice, Inbred C57BL ; Molecular Docking Simulation ; Nerve Tissue Proteins/metabolism* ; Rats ; Receptors, G-Protein-Coupled/metabolism* ; Receptors, Neuropeptide/therapeutic use*

OBJECTIVE: To evaluate the efficacy of iRoot BP Plus, a novel bioceramic material, and mineral trioxide aggregate (MTA) by comparing the clinical and radiographic results of pulpotomy in human primary molars, and to find out the influence factor. METHODS: Children who had at least one primary molar diagnosed as pulpitis, and received pulpotomy in the Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology from January, 2017 to December, 2018 were searched by the selection criteria in the Electronic Medical Record Database of Peking University School and Hospital of Stomatology. The molars with the pulp capping agents were iRoot BP Plus or MTA were included, and the molars with other pulp capping agents were excluded. Molars using iRoot BP Plus were selected into the experimental group (iRoot BP Plus group). Molars using MTA were selected into the control group (MTA group) using propensity score matching model (1 ∶ 1), considering the gender, age, tooth position, the title of clinician, pulp status, restoration and length of follow-up as the potential influence factor. Basic information, the relevant medical records including symptoms, clinical and radiographic examination before and after operation, intraoperative information and follow up examination records were collected. The success rates were compared and the influence factors were analyzed respectively. RESULTS: The study included 132 children, 178 molars (89 molars from each group) and the mean follow-up time was (462±99) days by December, 2019. In the iRoot BP Plus group, 9 molars failed whilst 6 molars failed in MTA group. The success rate was 89.9% and 93.3%, respectively, showing no significant difference between the two groups. Through Cox proportional risk model analysis, the success rate of iRoot BP Plus was significantly lower when the pulp status was poor, while other factors including age, tooth position, title of clinician and restoration were not found to be associated with the clinical outcome. CONCLUSION Pulpotomy of human primary molars with iRoot BP Plus has the same effectiveness with MTA. The pulp status is the main factor that influences the outcome of iRoot BP Plus. Further studies with larger sample sizes and longer follow-up terms are needed.
Child ; Humans ; Pulpotomy/methods* ; Pulp Capping and Pulpectomy Agents ; Pemetrexed ; Silicates/therapeutic use* ; Aluminum Compounds/therapeutic use* ; Calcium Compounds ; Oxides ; Molar/surgery* ; Drug Combinations ; Treatment Outcome ; Tooth, Deciduous

Epidemic diseases have caused huge harm to the society. Traditional Chinese medicine(TCM) has made great contributions to the prevention and treatment of them. It is of great reference value for fighting diseases and developing drugs to explore the medication law and mechanism of TCM under TCM theory. In this study, the relationship between the TCM theory of cold pestilence and modern epidemic diseases was investigated. Particularly, the the relationship of coronavirus disease 2019(COVID-19), severe acute respiratory syndrome(SARS), and influenza A(H1 N1) with the cold pestilence was identified and analyzed. The roles of TCM theory of cold pestilence in preventing and treating modern epidemic diseases were discussed. Then, through data mining and textual research, prescriptions for the treatment of cold pestilence were collected from major databases and relevant ancient books, and their medication laws were examined through analysis of high-frequency medicinals and medicinal pairs, association rules analysis, and cluster analysis. For example, the prescriptions with high confidence levels were identified: "Glycyrrhizae Radix et Rhizoma-Bupleuri Radix-Paeoniae Radix Alba" "Glycyrrhizae Radix et Rhizoma-Pinelliae Rhizoma-Bupleuri Radix", and TCM treatment methods with them were analyzed by clustering analysis to yield the medicinal combinations: "Zingiberis Rhizoma-Aconiti Lateralis Radix Praeparata-Ginseng Radix et Rhizoma" "Poria-Atractylodis Macrocephalae Rhizoma" "Cinnamomi Ramulus-Asari Radix et Rhizoma" "Citri Reticulatae Pericarpium-Perillae Folium" "Pinelliae Rhizoma-Magnoliae Officinalis Cortex-Atractylodis Rhizoma" "Paeoniae Radix Alba-Angelicae Sinensis Radix-Glycyrrhizae Radix et Rhizoma-Bupleuri Radix-Scutellariae Radix-Rhizoma Zingiberis Recens" "Ephedrae Herba-Armeniacae Semen Amarum-Gypsum Fibrosum" "Chuanxiong Rhizoma-Notopterygii Rhizoma et Radix-Angelicae Dahuricae Radix-Platycodonis Radix-Saposhnikoviae Radix". Then, according to the medication law for cold pestilence, the antiviral active components of medium-frequency and high-frequency medicinals were retrieved. It was found that these components exerted the antiviral effect by inhibiting virus replication, regulating virus proteins and antiviral signals, and suppressing protease activity. Based on network pharmacology, the mechanisms of the medicinals against severe acute respiratory syndrome coronavirus(SARS-CoV), 2019 novel coronavirus(2019-nCoV), and H1 N1 virus were explored. It was determined that the key targets were tumor necrosis factor(TNF), endothelial growth factor A(VEGFA), serum creatinine(SRC), epidermal growth factor receptor(EGFR), matrix metalloproteinase 9(MMP9), mitogen-activated protein kinase 14(MAPK14), and prostaglandin-endoperoxide synthase 2(PTGS2), which were involved the mitogen-activated protein kinase(MAPK) pathway, advanced glycation end-products(AGE)-receptor for AGE(RAGE) pathway, COVID-19 pathway, and mTOR pathway. This paper elucidated the medication law and mechanism of TCM for the prevention and treatment of epidemic diseases under the guidance of TCM theory of cold pestilence, in order to build a bridge between the theory and modern epidemic diseases and provide reference TCM methods for the prevention and treatment of modern epidemic diseases and ideas for the application of data mining to TCM treatment of modern diseases.
Aconitum ; Antiviral Agents ; COVID-19/epidemiology* ; Calcium Sulfate ; Communicable Disease Control ; Communicable Diseases/virology* ; Creatinine ; Cyclooxygenase 2 ; Drugs, Chinese Herbal/therapeutic use* ; Endothelial Growth Factors ; Epidemics/prevention & control* ; ErbB Receptors ; Humans ; Matrix Metalloproteinase 9 ; Medicine, Chinese Traditional ; Mitogen-Activated Protein Kinase 14 ; Pinellia ; SARS-CoV-2 ; TOR Serine-Threonine Kinases ; Tumor Necrosis Factors ; COVID-19 Drug Treatment