Application of anaerobic bacteria sensitive antibiotics in the preparation of drugs for treating preeclampsia

Abstract

The present application discloses the use of anaerobic-sensitive antibiotics in the preparation of drugs for the treatment of preeclampsia. Fusobacterium nucleatum is closely related to the clinical phenotype of preeclampsia, and has the effect of directly inducing the occurrence of preeclampsia, and clarifies the key pathogenic bacteria of preeclampsia. Taking Fusobacterium nucleatum as a target for preeclampsia, using anaerobic-sensitive antibiotics to target and eliminate this strain can improve the phenotypes of preeclampsia including hypertension, proteinuria, fetal growth restriction, and placental dysplasia.

Description

Technical Field

[0001] The present application relates to the field of medical microbiology technology, and in particular to the use of anaerobic-sensitive antibiotics in the preparation of drugs for treating preeclampsia. Background Art

[0002] Preeclampsia is a hypertensive disorder during pregnancy that affects multiple organ systems. Preeclampsia is defined as systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg after 20 weeks of pregnancy, accompanied by any of the following: urine protein ≥0.3 g / 24 h, or urine protein / creatinine ratio ≥0.3, or random urine protein ≥(+); no proteinuria but accompanied by any of the following maternal end-organ dysfunction: neurological complications, pulmonary edema, blood complications, acute kidney injury, liver involvement; or uterine placental dysfunction, etc. It is the main cause of death of pregnant women, fetuses, and newborns.

[0003] The intestinal flora is a general term for the microbial community living in the human intestine. It participates in regulating host metabolism, nutrition, immunity and other processes. Its role in intestinal and extraintestinal diseases has been widely studied. In normal pregnancy, the maternal intestinal flora is reprogrammed to adapt to maternal metabolic changes. Disturbances in the maternal intestinal flora can lead to complications during pregnancy and even affect the development of offspring. Previous studies have found that the intestinal flora of patients with preeclampsia is significantly different from that of normal pregnant women, with a significant decrease in bacterial α diversity, an increase in the abundance of harmful bacteria, and a decrease in the abundance of beneficial bacteria; transplanting feces from patients with preeclampsia into mice can induce the appearance of a preeclampsia phenotype in mice, indicating that intestinal flora disorders promote the occurrence of preeclampsia. However, the key pathogens that induce preeclampsia remain to be identified, and therapeutic technologies that use these key pathogens as intervention targets for preeclampsia also need to be developed. Summary of the invention

[0004] The purpose of the present application is to at least partially overcome the deficiencies of the prior art and provide the use of anaerobic-sensitive antibiotics in the preparation of drugs for treating preeclampsia.

[0005] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0006] Use of anaerobic bacteria-sensitive antibiotics in the preparation of drugs for treating preeclampsia.

[0007] Furthermore, the drug is used to treat preeclampsia induced by Fusobacterium nucleatum.

[0008] Specifically, the Fusobacterium nucleatum is Fusobacterium nucleatum, and its 16S rDNA sequence is shown in SEQ ID No.1.

[0009] Alternatively, the medicament is used for treating preeclampsia induced by Fusobacterium nucleatum damaging the intestinal barrier.

[0010] Further alternatively, the medicament is used to treat preeclampsia induced by Fusobacterium nucleatum migration to the placenta.

[0011] Further optionally, the drug is used to improve at least one of the following symptoms of preeclampsia: hypertension, increased urine protein, fetal development retardation, reduced embryo number, reduced fetal weight, and reduced placental weight.

[0012] Preferably, the anaerobic bacteria sensitive antibiotics include any one of the following: piperacillin, cefuroxime, latamoxef, clindamycin, imipenem, meropenem, and metronidazole.

[0013] Optionally, the drug is administered by injection or oral administration.

[0014] Compared with the prior art, this application has the following advantages:

[0015] (1) This application can treat pathogenic bacteria that induce preeclampsia, achieve the effect of targeted treatment, and help reduce additional medication;

[0016] (2) The anaerobic-sensitive antibiotics used in this application are easily available and have been maturely used in treating other diseases, and can be better applied in clinical practice;

[0017] (3) The symptoms that can be improved by this application are helpful to significantly relieve the patient's discomfort and restore uterine function, and it is expected to have significant therapeutic effects in clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the abundance of Fusobacterium and its species in the placenta of patients with preeclampsia;

[0019] Figure 2 To analyze the correlation between Fusobacterium nucleatum and clinical indicators of preeclampsia;

[0020] Figure 3 The colony growth morphology of Fusobacterium nucleatum (ATCC25586) cultured on Columbia blood agar plate;

[0021] Figure 4 This is the mass spectrometry identification result of Fusobacterium nucleatum;

[0022] Figure 5Fusobacterium nucleatum induces preeclampsia in mice;

[0023] Figure 6 The phenotype of Fusobacterium nucleatum is that it damages the intestinal barrier and translocates to the placenta.

[0024] Figure 7 The present application describes the use of metronidazole to target the elimination of Fusobacterium nucleatum followed by the preeclampsia phenotype. DETAILED DESCRIPTION

[0025] The present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0026] In order to identify the key pathogenic bacteria that induce preeclampsia and further confirm whether the pathogenic bacteria can be used as intervention targets, the following studies were conducted in this example:

[0027] Identification of key pathogens of preeclampsia:

[0028] Placentas of patients with preeclampsia were collected, and DNA from placental tissues was extracted using the QIAamp DNA Micro Kit. The abundance of Fusobacterium and its species in placental tissues was detected by qPCR. Fusobacterium species include Fusobacterium nucleatum, Fusobacterium necrophorum, Fusobacterium varium, Fusobacterium mortiferum, and Fusobacterium ulcerans. Figure 1 As shown in A, the positive rate of Fusobacterium in the placenta of patients with preeclampsia was 39.86%, which was significantly higher than the positive rate of Fusobacterium in the placenta of normal pregnant women (2.44%); among the five species of Fusobacterium, Fusobacterium nucleatum had the highest positive rate in the placenta of patients with preeclampsia, which was 35.66%.

[0029] Furthermore, specific fluorescent probes corresponding to the aforementioned strains were prepared, and FISH staining (fluorescence in situ hybridization) was performed on placental tissues of normal pregnant women and patients with preeclampsia. Figure 1 Results in B show that Fusobacterium nucleatum signals were detected in the placenta of patients with preeclampsia.

[0030] The correlation between Fusobacterium nucleatum and clinical indicators of preeclampsia was further investigated. Spearman correlation analysis was performed between the abundance of Fusobacterium nucleatum in the placenta and clinical indicators of preeclampsia. The indicators involved included blood pressure (SBP and DBP), neonatal weight (Neoweight), albumin level (ALB), alanine aminotransferase level (ALT), aspartate aminotransferase level (AST) and urine protein level (Urine protein). Figure 2 The results showed that the abundance of Fusobacterium nucleatum was positively correlated with systolic blood pressure (SBP) and urine protein level (Urine protein).

[0031] Cultivation and identification of Fusobacterium nucleatum:

[0032] To facilitate subsequent research, the Fusobacterium nucleatum (ATCC25586) involved in this application was purchased from ATCC (American Type Culture Collection), which is a subspecies of Fusobacterium nucleatum. After 16S rDNA sequencing and mass spectrometry identification, its sequence is shown in SEQ ID No. 1; the whole genome sequence refers to NCBI Reference Sequence: NZ_CP028101.1.

[0033] The strain was directly inoculated on Columbia blood agar plates and cultured anaerobically at 37°C for 24-48 hours to obtain growing colonies. Figure 3 As shown, the strain of the present application grows well on Columbia blood agar plates, with colonies having a diameter of 1-2 mm, an irregular circle, and slight protrusions.

[0034] like Figure 4 As shown, after the bacteria were pre-treated with protein or polypeptide extraction or purification reagents, mass spectrometry identification was performed, and the results showed that it was Fusobacterium nucleatum, that is, the strain was Fusobacterium nucleatum.

[0035] Fusobacterium nucleatum induces preeclampsia phenotype in mice:

[0036] Female C57BL / 6J mice aged 8 weeks were randomly divided into 3 groups, 8 mice in each group. The experiment was divided into blank control group (Control), bacteria negative control group (E. coli), and Fusobacterium nucleatum experimental group (F. nucleatum). The mice were pre-treated with ABX (antibiotic removal) gavage for 5 consecutive days (vancomycin, 100 mg / kg; neomycin, metronidazole, and ampicillin 200 mg / kg). After that, according to the group setting, PBS (phosphate buffer), E. coli, and F. nucleatum were gavaged for 1 week, and then caged. The plug was seen the next day, and the female mice with plugs were selected to continue gavage until 17.5 days of pregnancy. The blood pressure of mice was tested before caged, 0.5, 5.5, 9.5, 12.5, 15.5, and 17.5 days of pregnancy. The samples were dissected on the 17.5th day of pregnancy, and the feces, urine, placenta, and fetuses of the mice were collected. The ELISA method was used to detect the urine protein level of mice and the weight of placenta and offspring was measured. Figure 5 A, Blood pressure (SBP) of mice gavaged with Fusobacterium nucleatum (F.nucleatum) began to increase from 12.5 days of gestation and continued to 17.5 days of gestation. There was no significant change in blood pressure of mice in the blank control PBS group and the bacteria negative control group (E.coli). Figure 5 B, The urine protein level (Urine albumin / creatinine) of mice gavaged with Fusobacterium nucleatum increased; Figure 5 C. Oral administration of Fusobacterium nucleatum resulted in fetal developmental retardation, a decrease in the number of fetuses, a decrease in fetal weight, and a decrease in placenta weight. Figure 5 D, Oral administration of F. nucleatum resulted in an increase in the ratio of the placental labyrinth / junctional area, suggesting placental dysplasia. These phenotypes are consistent with the clinical manifestations of preeclampsia, indicating that F. nucleatum can induce preeclampsia phenotypes in mice.

[0037] Fusobacterium nucleatum damages the intestinal barrier of mice and migrates to the placenta:

[0038] The mouse colon tissue proteins were extracted and the expressions of three tight junction proteins ZO-1, Occludin and Claudin-4 of the intestinal barrier were detected by immunoblotting. Figure 6 The results of A showed that the expression of tight junction proteins in the colon of mice in the oral administration group of Fusobacterium nucleatum was significantly decreased. The abundance of Fusobacterium nucleatum in the feces and placenta of mice was detected by qPCR. Figure 6 B The results showed that the abundance of Fusobacterium nucleatum in the feces and placenta of mice with preeclampsia phenotype was significantly increased; FISH staining of the mouse placenta was performed using a Fusobacterium nucleatum-specific fluorescent probe. Figure 6 The results of C showed that positive signals of Fusobacterium nucleatum were detected in the placenta of mice in the oral administration group (indicated by the red arrow). These results indicate that Fusobacterium nucleatum can destroy the intestinal barrier of mice and then migrate to the placenta.

[0039] Anaerobic antibiotics improve preeclampsia induced by Fusobacterium nucleatum:

[0040] Anaerobic bacteria refer to a type of bacteria that can only grow under low oxygen partial pressure conditions, but cannot grow on the surface of solid culture medium under air and (or) 10% carbon dioxide concentration. According to their tolerance to oxygen, they can be divided into obligate anaerobes, microaerophilic anaerobes and facultative anaerobes. Commonly used drugs against anaerobic infections include: metronidazole, ornidazole, clindamycin, piperacillin-tazobactam / sulbactam, cefuroxime, latamoxef, moxifloxacin, imipenem, and meropenem.

[0041] This example uses the relatively broad-spectrum metronidazole as an example to verify the effect of intervening in preeclampsia with Fusobacterium nucleatum (F. nucleatum) as the target. The metronidazole clearance experiment is divided into three groups, namely the control group (Control), the Fusobacterium nucleatum group (Fn), and the Fusobacterium nucleatum + metronidazole intervention group (Fn+MZ). In the Fn group and the Fn+MZ group, mice were treated with Fusobacterium nucleatum by gavage according to the aforementioned process; the Fn+MZ group was additionally injected with metronidazole through the tail vein once every 3 days. The control group was gavaged with an equal volume of PBS. According to Figure 7 A, Compared with the Fn group, the blood pressure (SBP value) of pregnant mice in the Fn+MZ group was lower; Figure 7 B, Compared with the Fn group, the urine protein level (Urine albumin / creatinine) of pregnant mice in the Fn+MZ group was lower; Figure 7 C, Compared with the Fn group, the developmental retardation of the fetuses in the Fn+MZ group was alleviated, the number of fetuses increased, the fetal weight increased, and the placenta weight increased. Compared with the control group, there was no significant difference. Figure 7D, The structure and development of the placenta were improved in the Fn+MZ group. These results indicate that metronidazole treatment can improve the phenotype of preeclampsia induced by F. nucleatum, and other anaerobic-sensitive antibiotics also have the potential to improve the phenotype of preeclampsia induced by F. nucleatum.

[0042] The above results show that:

[0043] Fusobacterium nucleatum is closely related to the clinical phenotype of preeclampsia and has a direct role in inducing the occurrence of preeclampsia, identifying the key pathogenic bacteria of preeclampsia.

[0044] Targeting Fusobacterium nucleatum as a preeclampsia target and eliminating this strain using anaerobic-sensitive antibiotics can improve preeclampsia phenotypes including placental status.

[0045] Anaerobic-sensitive antibiotics are used to treat preeclampsia induced by Fusobacterium nucleatum. In clinical application, the route of administration can be determined based on the specific drug, and can be injection or oral administration.

[0046] Therefore, the use of the anaerobic sensitive antibiotic of the present application in the preparation of a drug for treating preeclampsia, preferably the drug is used to treat preeclampsia induced by Fusobacterium nucleatum. More specifically, the drug is used to treat preeclampsia induced by Fusobacterium nucleatum damaging the intestinal barrier; and / or, the drug is used to treat preeclampsia induced by Fusobacterium nucleatum migrating to the placenta. In other embodiments, the drug can also be used to at least partially improve the following phenotypes of preeclampsia: hypertension, increased proteinuria, embryonic retardation, reduced number of embryos, reduced fetal weight and reduced placental weight.

[0047] The above embodiments are preferred implementation modes of the present application, but are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present application should be equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. Use of an anaerobic-sensitive antibiotic in the preparation of a drug for treating preeclampsia, characterized in that: The drug uses Fusobacterium nucleatum as an intervention target and is used to treat preeclampsia induced by Fusobacterium nucleatum; the Fusobacterium nucleatum is Fusobacterium nucleatum, Its 16S rDNA sequence is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The medicine is used for treating preeclampsia induced by Fusobacterium nucleatum damaging the intestinal barrier.

3. The use according to claim 1, characterized in that The medicine is used for treating preeclampsia induced by Fusobacterium nucleatum migration to the placenta.

4. The use according to claim 1, characterized in that The drug is used to improve at least one of the following symptoms of preeclampsia: hypertension, increased urine protein, embryonic development retardation, reduced embryo number, reduced fetal weight, and reduced placental weight.

5. The use according to claim 1, characterized in that The anaerobic bacteria sensitive antibiotics include any one of the following: piperacillin, cefuroxime, latamoxef, clindamycin, imipenem, meropenem, and metronidazole.

6. The use according to claim 1, characterized in that The drug is administered by injection or oral administration.

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