**2. Materials and methods**

### **2.1. Materials**

**1. Introduction**

of an effective treatment for the condition.

ins such as histamines, ammonia, and NO<sup>2</sup>

of silicosis.

Silicosis is a pneumoconiosis disease caused by exposure to various forms of silica dust including crystalline silica and amorphous silica dust [1]. It is caused by the inhalation of fine silica particles, which are deposited in the lungs and ingested by macrophages. This triggers an immune response that stimulates the production of collagen around the particle, resulting in the formation of nodular lesions that obstruct the airways. Occupational exposure to silica dust and the resulting health problems are major public health issues in both developed and developing countries [2]. It was recently reported that 23 million workers in China have been exposed to crystalline silica dust, and that more than ten million workers in India are at risk of exposure [3, 4]. Similarly, more than 1.7 million workers in the USA and more than 3 million workers in Europe are likely to have been exposed to silica dust in the workplace [5, 6]. Silicosis has a range of adverse effects on health, including an increased susceptibility to tuberculosis, lung cancer, and pulmonary heart disease. These problems are exacerbated by the lack

108 Chinese Medical Therapies for Diabetes, Infertility, Silicosis and the Theoretical Basis

Kombucha is a drink made by fermenting sugar and tea extracts with kombucha. It is rich in acetic acid bacteria, yeast probiotics, acetic acid, and other organic acids that are beneficial to human health and can inhibit the growth of harmful bacteria. It has proven to be a good treatment for atrophic gastritis and gastric ulcer disease and can also help to regulate blood pressure and slow aging and to prevent and treat various diseases [7]. Kombucha contains two notable groups of microorganisms: *Gluconoacetobacter xylinus* (Xylinum) and yeasts. Xylinum secretes bacterial cellulose through holes in its cell walls. Interestingly, kombucha cultures produce bacterial cellulose more efficiently than cultures of Xylinum alone [8, 9]. The bacterial cellulose produced by Kampuchea cultures has a number of properties that make it potentially useful in medical applications, including good biocompatibility, thinness (the cellulose sheets are typically only 0.1 μm thick), and a high‐specific surface area. These properties mean that it functions as a nanoscale functional material with a defined three‐dimensional structure and a large number of surface‐exposed hydroxyl groups, which allow it to form strong noncovalent bonds with water and also a wide range of ions and organic compounds [10]. It has been demonstrated that bacterial cellulose efficiently adsorbs numerous toxic heavy metal ions, including Cu2+, Pb2+, Hg2+, and Cd2+. In addition, it adsorbs a range of nonmetallic tox-


esized that if kombucha was sprayed into lung tissues, the bacterial cellulose produced by the culture might adsorb dust and protein precipitates that would otherwise cause the symptoms

Chinese herbal kombucha preparations can be made by fermenting extracts of various plants with a Kampuchea culture. If the plants used in these preparations contain biologically active substances, the resulting Chinese herbal kombucha may combine the beneficial effects of the Kampuchea itself with those of the plants. A range of plants and herbs can be used for this purpose, including licorice, *Siratia grosvenori*, mangosteen, and chrysanthemum. Licorice is known to have numerous pharmacological effects and is widely used in traditional medicine. It contains a range of biologically active compounds, including glycyrrhizin, glycyrrhetinic

and formaldehyde [11–16]. We therefore hypoth-

Silica dust (99% particle diameter 0.5–10 μm with 80% of particles having diameters of 1–5 μm) was purchased from Sigma‐Aldrich. Tetrandrine was purchased from the Zhejiang Zhongyi Pharmaceutical Co., Ltd. A kit for measuring collagen (hydroxyproline) levels was purchased from the Nanjing Jiancheng Bioengineering Institute.

#### **2.2. Preparation of the Kombucha mixtures**

Kampuchea strains were purchased from the Beijing Institute of Food Research. A kombucha stock solution was prepared according to the method of Ai‐jun Lv [23]. The Chinese herbal extract was prepared by mixing tea (0.2% w/w), licorice (0.5% w/w), dried *Siratia grosvenori* fruit (0.5% w/w), and wild chrysanthemum (0.2% w/w) in water and boiling the resulting mixtures for 20 min. The boiled solution was then filtered, cooled to below 30°C, and mixed with a 20% dilution of the kombucha stock solution. After fermenting at 30°C for 2 weeks, the Chinese herbal kombucha solution was considered ready for use in experiments.

#### **2.3. Experimental animals**

Test animals were provided by the Hunan Slack Jingda experimental animal company (License number SCXK; Hunan). The animals used were specific pathogen‐free (SPF) Sprague‐Dawley (SD) rats that had been held under quarantine for 5 days.

#### **2.4. Tracheal injection of method for the contamination of silica dust**

A 50 mg/mL standard suspension of quartz (silica) dust in saline was prepared [25–27]. Prior to tracheal injection, samples of the stock solution were autoclaved and then mixed with mycillin (4000 U/mL). The experimental animals were then anaesthetized under sterile conditions and subjected to intratracheal injection with 1 mL of the sterile mycillin‐containing silica dust suspension in lungs.

#### **2.5. Experimental groups and treatments**

The 150 experimental animals weighed 180–220 g each and were randomly allocated to five different groups of 30 animals each, 15 males and 15 females (see **Table 1**).


**Table 1.** Experimental groups and treatments.

#### **2.6. Measurement of organ coefficients and the wet and dry weights of lung tissues**

At the end of the treatment period, the animals were killed by arterial bloodletting. Their hearts, livers, spleens, kidneys, and other organs and tissues were then immediately removed and weighed, and the organ coefficient for each organ was calculated (organ coefficient = organ weight/body weight × 100%). The trachea and lungs were then removed and separated, and the lungs were stripped of their connective tissue. The connective tissues were soaked in water, and the wet lung weight (M) was measured. A 0.5 g lung tissue sample was then immersed in acetone for 3 days for degreasing and then cut into pieces, baked in an oven at 105°C for 12 h, and weighed to determine its dry weight (m). The dry weight of the lung was then calculated as 2 m×M.

#### **2.7. Determination of total lung collagen (hydroxyproline)**

Lung homogenates were prepared, and their hydroxyproline content was determined using the chloramine‐T method, according to the instructions provided with the kit [27].

#### **2.8. Counting and classification of cells in lung lavage fluid**

The rats were killed by arterial bloodletting from the groin. The trachea was then removed, and a V‐shaped opening was made at the 1/3 point of the lower trachea. One end of a small plastic hose was fitted with an eight‐gauge needle, and the other end was inserted into the V‐shaped opening and ligated to the lung using fixed lines. 5 mL of saline was taken up in a syringe, which was then affixed to the needle on the end of the tubing. The saline was slowly injected into the alveoli and then slowly withdrawn to yield approximately 3 mL of recovered liquid. The cells within this recovered liquid were counted and classified under a microscope.

#### **2.9. Pathological analysis of lung tissues**

with a 20% dilution of the kombucha stock solution. After fermenting at 30°C for 2 weeks, the

Test animals were provided by the Hunan Slack Jingda experimental animal company (License number SCXK; Hunan). The animals used were specific pathogen‐free (SPF) Sprague‐Dawley

A 50 mg/mL standard suspension of quartz (silica) dust in saline was prepared [25–27]. Prior to tracheal injection, samples of the stock solution were autoclaved and then mixed with mycillin (4000 U/mL). The experimental animals were then anaesthetized under sterile conditions and subjected to intratracheal injection with 1 mL of the sterile mycillin‐containing silica

The 150 experimental animals weighed 180–220 g each and were randomly allocated to five

afternoon for 5 consecutive days per week, over a 4‐week period

afternoon for 5 consecutive days per week, over a 4‐week period

Kombucha treatment group Starting 4 days after being injected with silica, the rats were sprayed with non‐Chinese

for 5 consecutive days per week, over a 4‐week period Tetrandrine treatment group The rats were treated with tetrandrine by lavage. Each rat was dosed with 15 mg three

Positive control group Starting 4 days after being injected with silica, the rats were sprayed with a 1 g/L

Negative control group Synchronously operated with the positive control group, at the fourth day of instilled

in the afternoon for 5 consecutive days for 4 weeks

Starting 4 days after being injected with silica, the rats were sprayed with Chinese herbal kombucha. Each rat was sprayed 20 times in the morning and 20 times in the

herbal kombucha. Each rat was sprayed 20 times in the morning and in the afternoon

solution of NaCl. Each rat was sprayed 20 times in the morning and 20 times in the

with silica. Each rat was sprayed 20 times continuously once in the morning and once

**2.6. Measurement of organ coefficients and the wet and dry weights of lung tissues**

At the end of the treatment period, the animals were killed by arterial bloodletting. Their hearts, livers, spleens, kidneys, and other organs and tissues were then immediately removed and weighed, and the organ coefficient for each organ was calculated (organ coefficient = organ weight/body weight × 100%). The trachea and lungs were then removed and separated, and the lungs were stripped of their connective tissue. The connective tissues were

Chinese herbal kombucha solution was considered ready for use in experiments.

(SD) rats that had been held under quarantine for 5 days.

110 Chinese Medical Therapies for Diabetes, Infertility, Silicosis and the Theoretical Basis

**2.4. Tracheal injection of method for the contamination of silica dust**

different groups of 30 animals each, 15 males and 15 females (see **Table 1**).

times per week for 4 weeks

**2.3. Experimental animals**

dust suspension in lungs.

**2.5. Experimental groups and treatments**

**Group Treatment**

**Table 1.** Experimental groups and treatments.

Chinese herbal kombucha treatment group

First, the gross lung morphology was observed. The lung tissue was then fixed using formalin. Conventional paraffin sections were taken and stained with hematoxylin‐eosin. The stained sections were then examined using an optical microscope (Olympus BX43). Lung tissue pathological changes and nodules pathological were graded, and stained lung tissue collagen fiber and reticular fiber hyperplasia were observed with Model BX43, Olympus Optical microscopy. The results obtained were evaluated according to the diagnostic criteria for pneumoconiosis specified in the national occupational health standards of the People''s Republic of China (GBZ25‐2002).

#### **2.10. Determination of the free silica contents of whole lung samples using the pyrophosphate method**

Silica levels in lung samples were measured using the method for determining silica levels in dust samples described in Chinese national standard GBZ/T 192.4‐2007 ("Determination of dust in the air of workplace—Part 4: Content of free silica in dust") [28]. Fresh rat lung samples were degreased, dried, and then crushed. Samples of the crushed material (0.10 g) were then analyzed using the above method.
