Chinese Traditional Medicine NiuBeiXiaoHe (NBXH) Extracts Have the Function of Antituberculosis and Immune Recovery in BALB/c Mice

Background The Traditional Chinese Medicine NiuBeiXiaoHe (NBXH) is a valid antituberculosis (TB) prescription from the experience of clinical practice. However, the mechanism of NBXH extracts' immunotherapy has been poorly understood. Herein, the immunotherapeutic efficacy and the differentially expressed (DE) genes of NBXH extracts were evaluated and identified in BALB/c mice. Methods The total RNA was extracted from peripheral blood mononuclear cells, and the DE genes were identified by gene chip. The enrichment and signaling pathway analyses were performed using Gene Ontology (GO) and KEGG database. Results It was shown that the treatment of NBXH extracts (high dose) significantly reduced mycobacteria loads and histopathological lesions in mice infected by Mycobacterium tuberculosis and resulted in 3,454 DE upregulated genes and 3,594 downregulated DE genes. Furthermore, NBXH extracts killed mycobacteria by inhibiting the supply of necessary ingredients for their growth and proliferation. They restored the disordered immune microenvironments by up- or downregulating immune and inflammation-related pathways. Conclusions Taken together, NBXH extracts not only efficiently decreased the mycobacteria loads but also balanced the immune disorders in mice. These new findings provide a fresh perspective for elucidating the immunotherapeutic mechanism of NBXH extracts and pointed out the direction for improving the treatment efficacy of NBXH extracts.


Introduction
Tuberculosis (TB) is a global contagious disease that can affect almost any part of the body but is mainly found in the lungs. According to the Global TB report 2020 by the World Health Organization, it is estimated that there were 10.0 million TB patients in the world in 2019 [1]. TB is still a serious and growing public health problem. At present, TB is mainly treated with combination chemotherapy of western medicine. However, the treatment of western medicine has the following issues: First, the treatment of multidrug-resistant (MDR) TB and incredibly extensively drug-resistant (XDR) TB is complicated in the clinic, and requires combining second-line drugs and more than nine months of treatment course [2]. Second, antituberculosis chemical drugs usually have side effects; for example, severe liver injury was induced in about 10% of TB patients [3]. Third, the western medicine treatment on TB mainly emphasizes killing M. tuberculosis [4].
TB is not only a bacterium infectious disease but also an immune illness. TB's occurrence and development are closely related to immune dysfunction such as abnormal monocytemacrophage activation, Th1 immune response, the imbalance of Th1/Th2 immune response, and hypoimmunity [5]. Traditional Chinese Medicine (TCM) compound has multicomponent, multitarget, multipathway, and multieffect characteristics. It plays a synergistic effect with chemotherapy on killing M. tuberculosis, enhancing the patient's immune function, improving clinical symptoms and sputum negative rate, and increasing the curative percentage of TB [6]. Therefore, the treatment of TB with integrated traditional Chinese and western medicine is likely to open an effective way to extricate TB (especially MDR-TB and refractory TB) from a challenging situation.
The TCM compound NiuBeiXiaoHe (NBXH) is a valid anti-TB prescription from clinical practice experience [7]. NBXH is composed of six Chinese herbal medicines (Table 1 and Figure 1(a)). Bulbus Fritillariae Cirrhosae and Rhizoma Bletillae are the main components of NBXH, which have the functions of heat clearing, lung moistening, phlegm resolving, cough relieving, hemostasis, and detumescence [8][9][10]. Herba Houttuyniae, Radix Platycodonis, and Fructus Arctii are the auxiliary elements of NBXH, which have the beneficial effects of acting as an anti-inflammatory, reducing phlegm, and relieving cough as well as sore throat [11][12][13]. Glutinous rice is used as the adjuvant to invigorate the spleen, stomach, and lung [14]. The compound NBXH has been used to treat TB patients in the clinic for more than 20 years, and it could quickly eliminate symptoms such as chest pain, hemoptysis, low fever, night sweat, malnutrition, and fatigue; it could also improve the cure rate of TB [15]. Our previous studies found that NBXH extracts have some therapeutic effects on the TB mouse model by inhibiting or killing M. tuberculosis in vitro and in vivo, reducing lung lesions, and improving the general condition of mice [16,17].
However, the immunotherapeutic mechanism of NBXH extracts remains unclear. It was well known that isoniazid (INH) is the first-line drug for TB treatment in the clinic, and hence we chose it for positive control. Herein, we will compare the efficacy of NBXH extracts and INH in the treatment of tuberculosis, identify differentially expressed (DE) genes, and analyze potential target molecules and signaling pathways through gene chip technology and bioinformatics methods to explore the immunotherapeutic mechanism of NBXH extracts.

Mice and Ethics Statement.
Female BALB/c mice aged 6-8 weeks (17-19 g) were obtained from Vital River Laboratories (Beijing, China). All animal experiments were approved and directed by the Animal Ethical Committee of the 8th Medical Center of Chinese PLA General Hospital. Animal care and management were strictly carried out under the standards of Experimental Animal Regulation Ordinances formulated by the China National Science and Technology Commission. At the end of the experiments, the mice were euthanized with ketamine hydrochloride (2 mg/kg) and 2% xylazine (3 mg/kg) and then euthanized with cervical dislocation.
2.2. Mycobacterium tuberculosis Strain. Mycobacterium tuberculosis (H37Rv strain) was obtained from the Chinese Academy for Food and Drug Control and cultured on the Lowenstein-Jensen medium. Four weeks later, the mycobacterial colonies were converged to homogenize in saline (0.05% Tween 80) and then stored at -20°C. The colony-forming units (CFUs) of viable mycobacteria were determined by plating serial dilutions on the Lowenstein-Jensen medium.

The Preparation of NBXH Extracts.
The Chinese medicine compound NBXH was purchased from Guangdong Qifang Pharmaceutical Co., Ltd. (Guangzhou, China). The weight of each plant was listed in Table 1. The NBXH was extracted with water three times, and their filtrates were combined, concentrated, and then dried in a vacuum. All these steps were achieved by Guangdong Qifang Pharmaceutical Co., Ltd. The details of the NBXH extraction process can be found in our previous study [16].

The Preparation and Treatments of the TB-Infected
Mouse Model. A flowchart of the preparation and treatments of the TB-infected mouse model is shown in Figure 1(b). Fifty-three specific pathogen-free female BALB/c mice were challenged with 5 × 10 5 CFUs of M. tuberculosis strain  Three days before immunotherapy, 53 female BALB/c mice were infected by M. tuberculosis H37Rv strain with 5 × 10 5 CFUs per mouse. Then, three mice were sacrificed to determine whether the infection model had been successfully constructed. The remaining mice were randomly assigned to five groups and treated with water (negative control); INH (positive control); and NBXH-L, NBXH-M, or NBXH-H by intragastric administration five times in one week. In addition, as a blank control, ten normal mice without any treatment were kept in the same condition. After 91 days of initial immunotherapy, mice were sacrificed, and their lungs, livers, spleens, and blood were collected to evaluate the effect of immunotherapy or identify GE genes.
H37Rv suspension via tail vein injection. Three days after the challenge, three mice were randomly selected and sacrificed to determine whether the TB-infected mouse model was successfully constructed. Then, the remaining mice were divided into five groups (10 mice each group) and given distilled Water, INH (Shenyang Hongqi Pharmaceutical Co., Ltd., Liaoning, China), low-dose NBXH extracts (abbreviated as NBXH-L), medium-dose NBXH extracts (abbreviated as NBXH-M), or high-dose NBXH extracts (abbreviated as NBXH-H) by intragastric administration five times per week (except Saturday and Sunday), respectively. The experimental groups were briefly described as follows: negative control-TB-infected mice were treated with 0.5 ml distilled water; positive control-TB-infected mice were treated with 0.4 mg/0.5 ml INH; NBXH-L group-TB-infected mice were treated with 1.67 mg/0.5 ml NBXH extracts; NBXH-M group-TB-infected mice were treated with 3.35 mg/0.5 ml NBXH extracts; and NBXH-H group-TB-infected mice were treated with 6.7 mg/0.5 ml NBXH extracts. The dosage of NBXH extracts used by each mouse was calculated based on the practical clinical dose. Besides, the other ten healthy mice were left untreated and used as normal control (NC) for the DE gene identification. After thirteen weeks of the treatment, mice in the Water, INH, and NBXH groups were sacrificed, and the lung, liver, and spleen were collected to evaluate the immunotherapy efficacy. Furthermore, the blood samples were obtained and used to extract total RNA.
2.5. Evaluation of Immunotherapy Efficacy of the NBXH Extracts in TB Infected Mice. The weight changes of mice were recorded once a week since day zero in Figure 1(b). Ninety-one days past the first treatment, the body weight of each mouse was accurately weighed. Then, all mice were sacrificed. Their lungs and spleens were collected to observe gross pathological lesions (the evaluation criteria are listed in Table 2) and calculate organ coefficients (the ratio of organ weight to body weight). After that, the left lung lobe and the upper half of the spleen were homogenized in 3 ml normal saline, respectively. The tissue suspension was serially diluted at a ratio of 1 : 10, 1 : 100, and 1 : 1000 by using normal saline. Then, 0.1 ml diluted suspension was inoculated in duplicate on the modified Lowenstein-Jensen medium plates containing 0.1 μg/ml amphotericin B (North China Pharmaceutical Co., Ltd., Beijing, China) and incubated at 37°C for four weeks. M. tuberculosis colonies in each plate were counted and shown as CFUs per organ. Moreover, the right lung lobe of each mouse was fixed in 10% (vol/vol) neutral formalin and embedded in paraffin. The paraffin-embedded tissue sections were stained with hematoxylin/eosin (H&E) according to our previous study [18]. The histopathologic lesions of lung tissue were observed under a microscope, and the lesion area of the lung was calculated by Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA).
2.6. Extraction, Quantification, and Quality Analysis of Total RNA. Blood samples were collected from mice using an ethylenediaminetetraacetic acid dipotassium (EDTA-K2) anticoagulant tube. The peripheral blood mononuclear cells (PBMCs) were isolated from blood samples following a previous study [19].  [20]. Genomes) database to determine the role these DE genes play in biological pathways. Pathway analysis is a functional analysis mapping genes to KEGG pathways. The P value (EASEscore, Fisher-P value, or Hypergeometric-P value) denotes the significance of the pathway correlated to the conditions. The lower the P value, the more significant is the pathway (the recommended P value cut-off is 0.05) [21].
2.10. Statistical Analyses. Statistical analyses were performed using GraphPad Prism 8 software (San Diego, CA, USA). All the data were expressed as mean ± standard error of the mean (SEM). The significance of difference among groups was evaluated by one-way analysis of variance (ANOVA) or the Kruskal-Wallis test (nonparametric test) according to data normality and homogeneity of variances. The P value less than 0.05 indicated statistical significance. Gene expression profiling of mouse was analyzed by fold change.
The threshold used to screen up-or downregulated genes was fold change ≥ 2.

High-Dose NBXH Extracts
Significantly Reduced M. tuberculosis CFUs. Three days post infection, three mice were randomly selected to be sacrificed. Spot-like lesions in the lungs and slight enlargement of livers and spleens were observed, and bacillary counts in the lung and spleens were approximately 3.4 log10 and 4.1 log10, which suggested that the TB-infected mouse model had been developed successfully. After thirteen weeks' treatment, the mice in each group were killed, and their organs and blood samples were collected. Our data showed that mice in the NBXH-H or INH treatment group gained 14.93% or 15.62% more weight than initial data, while those in the Water, NBXH-L, or NBXH-M treatment group gained only 10.37%, 11.49%, or 5.83% in weight on 91 days after treatment (Figure 2(a)). Interestingly, we also found that the weight of mice in the NBXH-H or NBXH-L treatment group showed an upward trend from day 0 to day 51, while the weight of mice in other groups showed a decrease on the 9th day after the first immunotherapy.

High-Dose NBXH Extract-Treated Mice Had Less
Histopathological and Gross Pathological Lesions. Histopathological analysis was conducted in the mouse's right lung lobe from each group (Figure 3(a)). Noticeable pathological lesions were observed in mice's lungs in the Water, NBXH-L, or NBXH-M treatment group, such as thickened alveolar walls, a large number of inflammatory cell infiltration, inflammatory exudates in the alveoli, and infiltration of inflammatory cells around the vessel wall. In contrast, the pathological lesions in the lungs of mice treated with INH and NBXH-H were relatively minor, with only a slight thickening of the alveolar wall and a small amount of inflammatory cell infiltration. The lesion area of the lung was evaluated using the Image-Pro Plus software (Figure 3(b)). The results suggested that the lesion area of lungs collected from mice in the INH (P < 0:0001) or NBXH-H (P < 0:0001) treatment group was smaller than that in the Water treatment group, and the lesion area of lungs collected from mice in the NBXH-L (P < 0:0001) or NBXH-M (P = 0:0003) treatment group was bigger than that in the INH treatment group. The lesion area of lungs collected from mice in the NBXH-L (P < 0:0001) or NBXH-M (P = 0:0034) treatment group was significantly bigger than that in the NBXH-H treatment group. Moreover, the gross pathological analysis was also measured. Our data showed the following: (1) In the lungs (Figure 3(c)), the nodular lesion score of mice treated with INH (P < 0:0001) and NBXH-H (P = 0:0207) was significantly lower than that of mice treated with water, and the nodular lesion score of mice treated with INH was significantly lower than that of mice treated with NBXH-L (P = 0:0021). (2) In the spleens (Figure 3(d)), the nodular lesion score of mice treated with INH (P < 0:0001), NBXH-M (P = 0:0187), or NBXH-H (P = 0:0187) was significantly lower than that of mice treated with water, and the nodular lesion score of mice treated with INH was significantly lower than that of mice treated with NBXH-L (P = 0:0187). Journal of Immunology Research a distinguishable gene expression profiling between the groups. The significant DE genes between the two groups were identified through volcano plot filtering (fold change ≥ 2:0, P value ≤ 0.05) following our previous study [20]. The results revealed that 2,898 upregulated and 2,749 downregulated DE genes were identified between the Water treatment group and the NC group; 2,111 upregulated and 2,275 downregulated DE genes were identified between the INH treatment group and the Water treatment group; and 3,454 upregulated and 3,594 downregulated DE genes were identified between Organ coefficient (spleen) Figure 2: Immunotherapeutic efficacy of NBXH extracts. The original weight of each mouse was obtained on day 0. After the first immunotherapy, the weight change of each mouse was measured weekly (in (a), the error bar is represented by dotted lines). Ninety-one days after initial immunotherapy, all the mice were killed, and their left lobe of the lung (b) or spleen (b) was collected for counting CFUs, and organ coefficient of the lung (d) or spleen (e) was performed. All data are shown as means ± S:E:M: (n = 10). Differences were considered statistically significant at P < 0:05. * vs. Water: * P < 0:05; * * P < 0:01; * * * P < 0:001; * * * * P < 0:0001; # vs. INH: # P < 0:05; . Furthermore, the gross pathology of the lung and spleen was also determined, including the nodular lesion score in the lung (c) or spleen (d) according to the evaluation criteria in Table 2. Original magnification times: 40x. All data are shown as means ± S:E:M: (n = 10). Differences were considered statistically significant at P < 0:05. * vs. Water: * P < 0:05; * * P < 0:01; * * * P < 0:001; * * * * P < 0:0001; # vs. INH: ## P < 0:01; ### P < 0:001; #### P < 0:0001; § vs. NBXH-H: § § P < 0:01; § § § § P < 0:0001.   (Table S1). Surprisingly, we found that 23 genes were significantly downregulated or unchanged in mice infected with M. tuberculosis. However, all of them were upregulated considerably after INH or NBXH-H treatment (Table 3). In contrast, 18 genes were significantly upregulated or unchanged in mice infected with M. tuberculosis, whereas all were significantly downregulated after INH or NBXH-H treatment (Table 4).  Figures 5(a)-5(f), respectively. By analyzing these data, we unexpectedly found the following: (1) In M. tuberculosis-infected mice (Water treatment group), seven items in BP were significantly enriched with upregulated DE genes. In contrast, in mice treated with NBXH-H, these items were enriched considerably with  Journal of Immunology Research downregulated DE genes. These terms included gene expression, nucleic acid metabolic process, cellular macromolecule metabolic process, cellular nitrogen compound metabolic process, nucleobase-containing compound metabolic process, macromolecule metabolic process, and nitrogen compound metabolic process (Figure 6(a)). (2) Three terms in CC were significantly enriched with downregulated DE genes in mice infected with M. tuberculosis, while these were enriched with upregulated DE genes in mice treated with NBXH-H. These terms included the plasma membrane part, plasma membrane, and cell periphery (Figure 6(b)). (3) Five terms in MF were significantly enriched with upregulated DE genes in mice infected with M. tuberculosis, while these were enriched with downregulated DE genes in mice treated with NBXH-H. These terms included nucleotide binding, organic cyclic compound binding, nucleoside phosphate binding, small ion binding, and nucleic acid binding. Also, five terms in MF were significantly enriched with downregulated DE genes in mice infected with M. tuberculosis. In contrast, these were enriched with upregulated DE genes in mice treated with NBXH-H. These terms included cytoskeletal protein binding, enzyme regulator activity, catalytic activity, cation binding, metal ion binding, and ion binding (Figure 6(c)).

Identification of DE Genes before and after TB
Based on the above fact, we focused our analysis on the Water treatment group vs. the NC group and the NBXH-H treatment group vs. the Water treatment group. The top 10 terms of BP, CC, and MF ranked by the enrichment of DE genes are shown in Figure S1. After comparing the data obtained from the TB-infected mice and the NBXH-Htreated mice, we found that the number of upregulated or downregulated genes caused by NBXH-H treatment was more than that caused by TB infection in GO in terms of BP, CC, and MF. Moreover, the upregulated terms of BP, CC, and MF in TB-infected mice were downregulated after NBXH-H treatment, and the downregulated terms of BP, CC, and MF in TB-infected mice were upregulated after NBXH-H treatment.

Pathway Analyses of DE Genes before and after TB
Infection or INH and NBXH-H Treatment. The pathway analysis was carried out to identify the potential biological pathways by using the latest KEGG database. Our results indicated the following: (1) Compared with the NC group, 66 significant upregulated pathways or 78 significant downregulated pathways were identified in the Water treatment group (Table S2), the top 10 of which are shown in    (Table S2), the top 10 of which are shown in Figure 7 (Table S2), the top 10 of which were shown in Figure 7(c). In addition, we also analyzed the distribution of the top 10 signaling pathways in each group of mice (Figure 7(d)). It was found that four pathways (Spliceosome, RNA transport, Leishmaniasis, and allograft rejection) were upregulated in TB-infected mice but were downregulated after INH or NBXH-H treatment. Four pathways (Rap1 signaling pathway, vascular smooth muscle contraction, EFR calcium reabsorption, and pathway in cancer) were downregulated in TB-infected mice. In contrast, they were upregulated after INH or NBXH-H treatment.
Our results found that the Rap1 signaling pathway plays a vital role in NBXH immunotherapy. Therefore, we focused on this pathway. Rap1 is a small GTPase that plays a significant role in controlling cell-cell and cell-matrix interactions by regulating the functions of integrins and other adhesion molecules in various cell types. As shown in Figure 8, we found that 21 DE genes were significantly downregulated in TB-infected mice but were distinctly upregulated after     Rac, Integrin, p120, ERK, MEK3,6, PI3K, and Akt. Additionally, seven pathways were downregulated in TB-infected mice. Simultaneously, they were upregulated in NBXH-Htreated mice, including the RAS signaling pathway, calcium signaling pathway, regulation of actin cytoskeleton, focal adhesion, adherens junction, MAPK signaling pathway, and PI3K-Akt signaling pathway. In contrast, the T cell receptor signaling pathway was remarkably upregulated in TB-infected mice but was downregulated in NBXH-Htreated mice.
Furthermore, based on the concept that TB is an infectious disease and an immune disease, we compared the upregulated or downregulated pathways related to immune disease and inflammation among different groups (  including the TNF signaling pathway, regulation of actin cytoskeleton, the Ras signaling pathway, focal adhesion, the PI3K-Akt signaling pathway, the calcium signaling pathway, the MAPK signaling pathway, and the TGF-beta signaling pathway. Four pathways were upregulated after TB infection and downregulated after NBXH-H treatment, including the NF-kappa B signaling pathway, the Toll-like receptor signaling pathway, intestinal immune network for IgA production, and the T cell receptor signaling pathway.

Discussion
TCM has been practiced in China for thousands of years and has saved countless lives. With the rapid development of modern medicine, a growing number of studies focused on understanding and applying Western medicine methods and techniques to study the pharmacology and efficacy of TCM-derived herbs [22]. As an ancient infectious disease, TB has accompanied humans from the Stone Age till today [23]. TB has been studied for 120 years, but the history of Chinese people using TCM to treat TB can be traced back 500 years ago. To date, several herbal medicines have been demonstrated to have immunotherapeutic efficacies on M. tuberculosis infection in animal models, including Allicin [24], Baicalin [25], Yokuinin [26], Mao-Bushi-Saishin-To [26], Astragalus mongholicus Bunge [27], Paeonia lactiflora Pall. [27], Curcuma longa L. [28], and a water extract of Ranunculus ternatus Thunb., Sophora flavescens Aiton, Prunella vulgaris L., and Stellera chamaejasme L. [29].
In a previous study, we compared the differences between three NBXH extraction methods on a mouse model. We found that the extracts from the three methods all had significant therapeutic effects against TB infection [16]. In the current study, we assessed the immunotherapeutic efficacy of the NBXH extracts (three doses) or INH in TB-infected BALB/c mice. Our results indicated that compared with the mice treated with water, the high-dose NBXH extracts or INHtreated mice gained more weight, had a greater number of CFUs and a significantly lower organ coefficient in the lung, and the pathological lesions of the lungs were lighter. It is noteworthy that INH had a more vital antituberculosis ability than NBXH extracts in both lungs and spleens of mice, indicating that its mechanism of killing intracellular mycobacteria might be different from NBXH extracts. As a prodrug, INH must be activated and catalyzed to the isonicotinic acyl radical by a bacterial catalase-peroxidase enzyme named KatG and coupled with NADH to form the nicotinoy1-NAD complex, which can bind tightly to InhA to inhibit the synthesis of the mycobacterial cell wall [4].
Although INH's action mechanism has been studied relatively clearly, that of NBXH extracts is poorly understood. Based on our previous research, we have proposed the following hypothesis: NBXH extracts would cause differential expression of genes in mice infected with TB. This might provide a new perspective for elucidating the antituberculosis mechanism of NBXH extracts. Encouragingly, 3   M. tuberculosis infection leads to increased intracellular calcium influx or release of calcium ions from intracellular calcium pools to activate intracellular calcium signaling pathways, which will activate the expression of genes encoding intracellular antiinflammatory-related protein and an immune protective related protein, enhance the killing and phagocytic capacity of immune cells such as macrophages, and ultimately clear M. tuberculosis in vivo.

mmu04010
MAPK signaling pathway -2.125314 NS +1.443387 The mitogen-activated protein kinase (MAPK) cascade is a highly conserved module that is involved in cell proliferation, differentiation, and migration. Activation of the MAPK signaling pathway promotes apoptosis, which facilitates host clearance of M. tuberculosis in infected cells.
mmu04350 TGF-beta signaling pathway -1.700798 NS +1.326878 The transforming growth factor-beta (TGF-beta) family members are structurally related secreted cytokines found in species ranging from worms and insects to mammals. A wide spectrum of cellular functions such as proliferation, apoptosis, differentiation, and migration are regulated by TGF-beta family members.