*Opioid Tolerance*

Opioids are known for their anti-nociceptive, anti-tussive and anti-diarrheal properties. They are the major drugs used in cancer and post-surgical pain treatment [133], although their severe side effects, such as tolerance, dependence, emesis, or constipation, lead to significant restrictions in their use [12]. GI symptoms associated with these drugs are known as opioid bowel dysfunction (OBD) and are the result of the stimulation of opioid receptors in the GI tract [134]. The research shows that chronic use of opioids may result in dysbiosis [12,135], damage to the gut barrier, bacterial translocation, and secretion of pro-inflammatory factors. Opioid tolerance was associated with a lack of *Bifidobacteria* and *Lactobacillaceae* in mice [12,25]. The enteric glia are responsible for the proper functioning of the GI tract [12]. Furthermore, they are also relevant to the development of the ectypal inflammatory reaction to long-term use of opioid drugs [136]. The bacterial product bacterial lipopolysaccharide (LPS) was reported to be associated with the production of proinflammatory cytokines during long-term opioid treatment [136]. Due to the chronic use of morphine, we can observe increased activity in enteric glia of the P2X receptor [12,136], a calcium-permeable ion channel activated by ATP and associated with cytokine secretion by enteric glia [25]. This leads to an enhanced inflammatory reaction [25]. LPS is also related to the intensified expression of connexin 43 (Cx43), a gap junction protein that mediates the secretion of ATP [136]. Cx43 can be blocked by non-specific connexin inhibitor (CBX), which results in a decreased inflammatory response [136].

Another study showed that administration of broad-spectrum antibiotics prevents GI side effects and tolerance to opioid-related drugs with long-term use of morphine [137]. Analgesic tolerance can be avoided by oral vancomycin due to its active properties against Gram-positive bacteria, the translocation of which is significant in the tolerance process [12,25,137]. Furthermore, germ-free mice have reduced morphine tolerance, which can be reclaimed by gut microbiome reconstitution [138]. In addition, opioid tolerance can be a result of the inactivation of tetrodotoxin-resistant (TTX-R) Na+ channels in dorsal root ganglia (DGR) neurons, which can be reversed by oral vancomycin administration [139]. In conclusion, the above-described studies prove the importance of the gut microbiota in opioid tolerance occurrence. They show the role of the gut flora in the genesis of morphine tolerance and indicate how the side effects of opioid drug use may enhance the entire process.

#### **6. The Gut Microbiota as a Therapeutic Target in Chronic Pain**
